Receptor activator of NF-kappaB

ABSTRACT

Isolated receptors, DNAs encoding such receptors, and pharmaceutical compositions made therefrom, are disclosed. The isolated receptors can be used to regulate an immune response. The receptors are also useful in screening for inhibitors thereof.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

[0001] This application is a continuation-in-part of U.S. Ser. No.60/064,671, filed Oct. 14, 1997, and a continuation in part of U.S. Ser.No. 08/813,509, filed Mar. 7, 1997, and a continuation-in part of U.S.Ser. No. 60/059,978, filed Dec. 23, 1996.

TECHNICAL FIELD OF THE INVENTION

[0002] The present invention relates generally to the field of cytokinereceptors, and more specifically to cytokine receptor/ligand pairshaving immunoregulatory activity.

BACKGROUND OF THE INVENTION

[0003] Efficient functioning of the immune system requires a finebalance between cell proliferation and differentiation and cell death,to ensure that the immune system is capable of reacting to foreign, butnot self antigens. Integral to the process of regulating the immune andinflammatory response are various members of the Tumor Necrosis Factor(TNF) Receptor/Nerve Growth Factor Receptor superfamily (Smith et al.,Science 248:1019; 1990). This family of receptors includes two differentTNF receptors (Type I and Type II; Smith et al., supra; and Schall etal., Cell 61:361, 1990), nerve growth factor receptor (Johnson et al.,Cell 47:545, 1986), B cell antigen CD40 (Stamenkovic et al., EMBO J.8:1403, 1989), CD27 (Camerini et al., J. Immunol. 147:3165, 1991), CD30(Durkop et al., Cell 68:421, 1992), T cell antigen OX40 (Mallett et al.,EMBO J. 9:1063, 1990), human Fas antigen (Itoh et al., Cell 66:233,1991), murine 4-1BB receptor (Kwon et al., Proc. Natl. Acad. Sci. USA86:1963, 1989) and a receptor referred to as Apoptosis-Inducing Receptor(AIR; U.S. Ser. No. 08/720,864, filed Oct. 4, 1996).

[0004] CD40 is a receptor present on B lymphocytes, epithelial cells andsome carcinoma cell lines that interacts with a ligand found onactivated T cells, CD40L (U.S. Ser. No. 08/249,189, filed May 24, 1994).The interaction of this ligand/receptor pair is essential for both thecellular and humoral immune response. Signal transduction via CD40 ismediated through the association of the cytoplasmic domain of thismolecule with members of the TNF receptor-associated factors (TRAFs;Baker and Reddy, Oncogene 12:1, 1996). It has recently been found thatmice that are defective in TRAF3 expression due to a targeted disruptionin the gene encoding TRAF3 appear normal at birth but developprogressive hypoglycemia and depletion of peripheral white cells, anddie by about ten days of age (Xu et al., Immunity 5:407, 1996). Theimmune responses of chimeric mice reconstituted with TRAF3^(−/−) fetalliver cells resemble those of CD40-deficient mice, although TRAF3^(−/−)B cells appear to be functionally normal.

[0005] The critical role of TRAF3 in signal transduction may be in itsinteraction with one of the other members of the TNF receptorsuperfamily, for example, CD30 or CD27, which are present on T cells.Alternatively, there may be other, as yet unidentified members of thisfamily of receptors that interact with TRAF3 and play an important rolein postnatal development as well as in the development of a competentimmune system. Identifying additional members of the TNF receptorsuperfamily would provide an additional means of regulating the immuneand inflammatory response, as well as potentially providing furtherinsight into post-natal development in mammals.

SUMMARY OF THE INVENTION

[0006] The present invention provides a novel receptor, referred to asRANK (for receptor activator of NF-κB), that is a member of the TNFreceptor superfamily. RANK is a Type I transmembrane protein having 616amino acid residues that interacts with TRAF3. Triggering of RANK byover-expression, co-expression of RANK and membrane bound RANK ligand(RANKL), and with addition of soluble RANKL or agonistic antibodies toRANK results in the upregulation of the transcription factor NF-κB, aubiquitous transcription factor that is most extensively utilized incells of the immune system.

[0007] Soluble forms of the receptor can be prepared and used tointerfere with signal transduction through membrane-bound RANK, andhence upregulation of NF-κB; accordingly, pharmaceutical compositionscomprising soluble forms of the novel receptor are also provided.Inhibition of NF-κB by RANK antagonists may be useful in amelioratingnegative effects of an inflammatory response that result from triggeringof RANK, for example in treating toxic shock or sepsis,graft-versus-host reactions, or acute inflammatory reactions. Solubleforms of the receptor will also be useful in vitro to screen foragonists or antagonists of RANK activity.

[0008] The cytoplasmic domain of RANK will be useful in developingassays for inhibitors of signal transduction, for example, for screeningfor molecules that inhibit interaction of RANK with TRAF2 or TRAF3.Deleted forms and fusion proteins comprising the novel receptor are alsodisclosed.

[0009] The present invention also identifies a counterstructure, orligand, for RANK, referred to as RANKL. RANKL is a Type 2 transmembraneprotein with an intracellular domain of less than about 50 amino acids,a transmembrane domain and an extracellular domain of from about 240 to250 amino acids. Similar to other members of the TNF family to which itbelongs, RANKL has a ‘spacer’ region between the transmembrane domainand the receptor binding domain that is not necessary for receptorbinding. Accordingly, soluble forms of RANKL can comprise the entireextracellular domain or fragments thereof that include the receptorbinding region.

[0010] These and other aspects of the present invention will becomeevident upon reference to the following detailed description of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011]FIG. 1 demonstrates the influence of RANK.Fc and hRANKL onactivated T cell growth. Human peripheral blood T cells were cultured asdescribed in Example 12; viable T cell recovery was determined bytriplicate trypan blue countings.

[0012]FIG. 2 illustrates the ability of RANKL to induce human DC clusterformation. Functionally mature dendritic cells (DC) were generated invitro from CD34⁺ bone marrow (BM) progenitors and cultured as describedin Example 13. CD1a⁺ DC were cultured in a cytokine cocktail alone(upper left panel), in cocktail plus CD40L (upper right), RANKL (lowerleft), or heat inactivated (ΔH) RANKL, and then photographed using aninversion microscope.

[0013]FIG. 3 demonstrates that RANKL enhances DC allo-stimulatorycapacity. Allogeneic T cells were incubated with varying numbers ofirradiated DC cultured as described in Example 13. The cultures werepulsed with [³H]-thymidine and the cells harvested onto glass fibersheets for counting. Values represent the mean ±standard deviation (SD)of triplicate cultures.

[0014]FIG. 4 presents an alignment of human RANK with other TNFR familymembers in the region of structurally conserved extracellularcysteine-rich pseudorepeats. Predicted disulfide linkages (DS1-DS3) areindicated. RANK and CD40 contain identical amino acid substitutions (C^H, C^ G) eliminating DS2 in the second pseudorepeat.

[0015]FIG. 5 presents an alignment of human RANKL with other TNF familymembers.

DETAILED DESCRIPTION OF THE INVENTION

[0016] A novel partial cDNA insert with a predicted open reading framehaving some similarity to CD40 was identified in a database containingsequence information from cDNAs generated from human bone marrow-deriveddendritic cells (DC). The insert was used to hybridize to colony blotsgenerated from a DC cDNA library containing full-length cDNAs. Severalcolony hybridizations were performed, and two clones (SEQ ID NOs:1 and3) were isolated. SEQ ID NO:5 shows the nucleotide and amino acidsequence of a predicted full-length protein based on alignment of theoverlapping sequences of SEQ ID NOs: 1 and 3.

[0017] RANK is a member of the TNF receptor superfamily; it most closelyresembles CD40 in the extracellular region. Similar to CD40, RANKassociates with TRAF2 and TRAF3 (as determined by co-immunoprecipitationassays substantially as described by Rothe et al., Cell 83:1243, 1995).TRAFs are critically important in the regulation of the immune andinflammatory response. Through their association with various members ofthe TNF receptor superfamily, a signal is transduced to a cell. Thatsignal results in the proliferation, differentiation or apoptosis of thecell, depending on which receptor(s) is/are triggered and which TRAF(s)associate with the receptor(s); different signals can be transduced to acell via coordination of various signaling events. Thus, a signaltransduced through one member of this family may be proliferative,differentiative or apoptotic, depending on other signals beingtransduced to the cell, and/or the state of differentiation of the cell.Such exquisite regulation of this proliferative/apoptotic pathway isnecessary to develop and maintain protection against pathogens;imbalances can result in autoimmune disease.

[0018] RANK is expressed on epithelial cells, some B cell lines, and onactivated T cells. However, its expression on activated T cells is late,about four days after activation. This time course of expressioncoincides with the expression of Fas, a known agent of apoptosis. RANKmay act as an anti-apoptotic signal, rescuing cells that express RANKfrom apoptosis as CD40 is known to do. Alternatively, RANK may confirman apoptotic signal under the appropriate circumstances, again similarto CD40. RANK and its ligand are likely to play an integral role inregulation of the immune and inflammatory response.

[0019] Moreover, the post-natal lethality of mice having a targeteddisruption of the TRAF3 gene demonstrates the importance of thismolecule not only in the immune response but in development. Theisolation of RANK, as a protein that associates with TRAF3, and itsligand will allow further definition of this signaling pathway, anddevelopment of diagnostic and therapeutic modalities for use in the areaof autoimmnune and/or inflammatory disease.

[0020] DNAs, Proteins and Analogs

[0021] The present invention provides isolated RANK polypeptides andanalogs (or muteins) thereof having an activity exhibited by the nativemolecule (i.e, RANK muteins that bind specifically to a RANK ligandexpressed on cells or immobilized on a surface or to RANK-specificantibodies; soluble forms thereof that inhibit RANK ligand-inducedsignaling through RANK). Such proteins are substantially free ofcontaminating endogenous materials and, optionally, without associatednative-pattern glycosylation. Derivatives of RANK within the scope ofthe invention also include various structural forms of the primaryproteins which retain biological activity. Due to the presence ofionizable amino and carboxyl groups, for example, a RANK protein may bein the form of acidic or basic salts, or may be in neutral form.Individual amino acid residues may also be modified by oxidation orreduction. The primary amino acid structure may be modified by formingcovalent or aggregative conjugates with other chemical moieties, such asglycosyl groups, lipids, phosphate, acetyl groups and the like, or bycreating amino acid sequence mutants. Covalent derivatives are preparedby linking particular functional groups to amino acid side chains or atthe N- or C-termini.

[0022] Derivatives of RANK may also be obtained by the action ofcross-linking agents, such as M-maleimidobenzoyl succinimide ester andN-hydroxysuccinimide, at cysteine and lysine residues. The inventiveproteins may also be covalently bound through reactive side groups tovarious insoluble substrates, such as cyanogen bromide-activated,bisoxirane-activated, carbonyldiimidazole-activated or tosyl-activatedagarose structures, or by adsorbing to polyolefin surfaces (with orwithout glutaraldehyde cross-linking). Once bound to a substrate, theproteins may be used to selectively bind (for purposes of assay orpurification) antibodies raised against the proteins or against otherproteins which are similar to RANK or RANKL, as well as other proteinsthat bind RANK or RANKL or homologs thereof.

[0023] Soluble forms of RANK are also within the scope of the invention.The nucleotide and predicted amino acid sequence of the RANK is shown inSEQ ID NOs:1 through 6. Computer analysis indicated that the protein hasan N-terminal signal peptide; the predicted cleavage site followsresidue 24. Those skilled in the art will recognize that the actualcleavage site may be different than that predicted by computer analysis.Thus, the N-terminal amino acid of the cleaved peptide is expected to bewithin about five amino acids on either side of the predicted, preferredcleavage site following residue 24. Moreover a soluble form beginningwith amino acid 33 was prepared; this soluble form bound RANKL. Thesignal peptide is predicted to be followed by a 188 amino acidextracellular domain, a 21 amino acid transmembrane domain, and a 383amino acid cytoplasmic tail.

[0024] Soluble RANK comprises the signal peptide and the extracellulardomain (residues 1 to 213 of SEQ ID NO:6) or a fragment thereof.Alternatively, a different signal peptide can be substituted for thenative leader, beginning with residue 1 and continuing through a residueselected from the group consisting of amino acids 24 through 33(inclusive) of SEQ ID NO:6. Moreover, fragments of the extracellulardomain will also provide soluble forms of RANK. Fragments can beprepared using known techniques to isolate a desired portion of theextracellular region, and can be prepared, for example, by comparing theextracellular region with those of other members of the TNFR family andselecting forms similar to those prepared for other family members.Alternatively, unique restriction sites or PCR techniques that are knownin the art can be used to prepare numerous truncated forms which can beexpressed and analyzed for activity.

[0025] Fragments can be prepared using known techniques to isolate adesired portion of the extracellular region, and can be prepared, forexample, by comparing the extracellular region with those of othermembers of the TNFR family (of which RANK is a member) and selectingforms similar to those prepared for other family members. Alternatively,unique restriction sites or PCR techniques that are known in the art canbe used to prepare numerous truncated forms which can be expressed andanalyzed for activity.

[0026] Other derivatives of the RANK proteins within the scope of thisinvention include covalent or aggregative conjugates of the proteins ortheir fragments with other proteins or polypeptides, such as bysynthesis in recombinant culture as N-terminal or C-terminal fusions.For example, the conjugated peptide may be a signal (or leader)polypeptide sequence at the N-terminal region of the protein whichco-translationally or post-translationally directs transfer of theprotein from its site of synthesis to its site of function inside oroutside of the cell membrane or wall (e.g., the yeast α-factor leader).

[0027] Protein fusions can comprise peptides added to facilitatepurification or identification of RANK proteins and homologs (e.g.,poly-His). The amino acid sequence of the inventive proteins can also belinked to an identification peptide such as that described by Hopp etal., Bio/Technology 6:1204 (1988). Such a highly antigenic peptideprovides an epitope reversibly bound by a specific monoclonal antibody,enabling rapid assay and facile purification of expressed recombinantprotein. The sequence of Hopp et al. is also specifically cleaved bybovine mucosal enterokinase, allowing removal of the peptide from thepurified protein. Fusion proteins capped with such peptides may also beresistant to intracellular degradation in E. coli.

[0028] Fusion proteins further comprise the amino acid sequence of aRANK linked to an immunoglobulin Fc region. An exemplary Fc region is ahuman IgG₁ having a nucleotide an amino acid sequence set forth in SEQID NO:8. Fragments of an Fc region may also be used, as can Fc muteins.For example, certain residues within the hinge region of an Fc regionare critical for high affinity binding to FcγRI. Canfield and Morrison(J. Exp. Med. 173:1483; 1991) reported that Leu₍₂₃₄₎ and Leu₍₂₃₅₎ werecritical to high affinity binding of IgG₃ to FcγRI present on U937cells. Similar results were obtained by Lund et al. (J. Immunol.147:2657, 1991; Molecular Immunol. 29:53, 1991). Such mutations, aloneor in combination, can be made in an IgG₁ Fc region to decrease theaffinity of IgG₁ for FcR. Depending on the portion of the Fc regionused, a fusion protein may be expressed as a dimer, through formation ofinterchain disulfide bonds. If the fusion proteins are made with bothheavy and light chains of an antibody, it is possible to form a proteinoligomer with as many as four RANK regions.

[0029] In another embodiment, RANK proteins further comprise anoligomerizing peptide such as a leucine zipper domain. Leucine zipperswere originally identified in several DNA-binding proteins (Landschulzet al., Science 240:1759, 1988). Leucine zipper domain is a term used torefer to a conserved peptide domain present in these (and other)proteins, which is responsible for dimerization of the proteins. Theleucine zipper domain (also referred to herein as an oligomerizing, oroligomer-forming, domain) comprises a repetitive heptad repeat, withfour or five leucine residues interspersed with other amino acids.Examples of leucine zipper domains are those found in the yeasttranscription factor GCN4 and a heat-stable DNA-binding protein found inrat liver (C/EBP; Landschulz et al., Science 243:1681, 1989). Twonuclear transforming proteins, fos and jun, also exhibit leucine zipperdomains, as does the gene product of the murine proto-oncogene, c-myc(Landschulz et al., Science 240:1759, 1988). The products of the nuclearoncogenes fos and jun comprise leucine zipper domains preferentiallyform a heterodimer (O'Shea et al., Science 245:646, 1989; Turner andTjian, Science 243:1689, 1989). The leucine zipper domain is necessaryfor biological activity (DNA binding) in these proteins.

[0030] The fusogenic proteins of several different viruses, includingparamyxovirus, coronavirus, measles virus and many retroviruses, alsopossess leucine zipper domains (Buckland and Wild, Nature 338:547, 1989;Britton, Nature 353:394, 1991; Delwart and Mosialos, AIDS Research andHuman Retroviruses 6:703, 1990). The leucine zipper domains in thesefusogenic viral proteins are near the transmembrane region of theproteins; it has been suggested that the leucine zipper domains couldcontribute to the oligomeric structure of the fusogenic proteins.Oligomerization of fusogenic viral proteins is involved in fusion poreformation (Spruce et al, Proc. Natl. Acad. Sci. U.S.A. 88:3523, 1991).Leucine zipper domains have also been recently reported to play a rolein oligomerization of heat-shock transcription factors (Rabindran etal., Science 259:230, 1993).

[0031] Leucine zipper domains fold as short, parallel coiled coils.(O'Shea et al., Science 254:539; 1991). The general architecture of theparallel coiled coil has been well characterized, with a“knobs-into-holes” packing as proposed by Crick in 1953 (ActaCrystallogr. 6:689). The dimer formed by a leucine zipper domain isstabilized by the heptad repeat, designated (abcdefg)_(n) according tothe notation of McLachlan and Stewart (J. Mol. Biol. 98:293; 1975), inwhich residues a and d are generally hydrophobic residues, with d beinga leucine, which line up on the same face of a helix. Oppositely-chargedresidues commonly occur at positions g and e. Thus, in a parallel coiledcoil formed from two helical leucine zipper domains, the “knobs” formedby the hydrophobic side chains of the first helix are packed into the“holes” formed between the side chains of the second helix.

[0032] The leucine residues at position d contribute large hydrophobicstabilization energies, and are important for dimer formation (Krysteket al., Int. J. Peptide Res. 38:229, 1991). Lovejoy et al. recentlyreported the synthesis of a triple-stranded α-helical bundle in whichthe helices run up-up-down (Science 259:1288, 1993). Their studiesconfirmed that hydrophobic stabilization energy provides the maindriving force for the formation of coiled coils from helical monomers.These studies also indicate that electrostatic interactions contributeto the stoichiometry and geometry of coiled coils.

[0033] Several studies have indicated that conservative amino acids maybe substituted for individual leucine residues with minimal decrease inthe ability to dimerize; multiple changes, however, usually result inloss of this ability (Landschulz et al., Science 243:1681, 1989; Turnerand Tjian, Science 243:1689, 1989; Hu et al., Science 250:1400, 1990).van Heekeren et al. reported that a number of different amino residuescan be substituted for the leucine residues in the leucine zipper domainof GCN4, and further found that some GCN4 proteins containing twoleucine substitutions were weakly active (Nucl. Acids Res. 20:3721,1992). Mutation of the first and second heptadic leucines of the leucinezipper domain of the measles virus fusion protein (MVF) did not affectsyncytium formation (a measure of virally-induced cell fusion); however,mutation of all four leucine residues prevented fusion completely(Buckland et al., J. Gen. Virol. 73:1703, 1992). None of the mutationsaffected the ability of MVF to form a tetramer.

[0034] Amino acid substitutions in the a and d residues of a syntheticpeptide representing the GCN4 leucine zipper domain have been found tochange the oligomerization properties of the leucine zipper domain(Alber, Sixth Symposium of the Protein Society, San Diego, Calif.). Whenall residues at position a are changed to isoleucine, the leucine zipperstill forms a parallel dimer. When, in addition to this change, allleucine residues at position d are also changed to isoleucine, theresultant peptide spontaneously forms a trimeric parallel coiled coil insolution. Substituting all amino acids at position d with isoleucine andat position a with leucine results in a peptide that tetramerizes.Peptides containing these substitutions are still referred to as leucinezipper domains.

[0035] Also included within the scope of the invention are fragments orderivatives of the intracellular domain of RANK. Such fragments areprepared by any of the herein-mentioned techniques, and include peptidesthat are identical to the cytoplasmic domain of RANK as shown in SEQ IDNO:5, or of murine RANK as shown in SEQ ID NO:15, and those thatcomprise a portion of the cytoplasmic region. All techniques used inpreparing soluble forms may also be used in preparing fragments oranalogs of the cytoplasmic domain (i.e., RT-PCR techniques or use ofselected restriction enzymes to prepare truncations). DNAs encoding allor a fragment of the intracytoplasmic domain will be useful inidentifying other proteins that are associated with RANK signalling, forexample using the immunoprecipitation techniques described herein, oranother technique such as a yeast two-hybrid system (Rothe et al.,supra).

[0036] The present invention also includes RANK with or withoutassociated native-pattern glycosylation. Proteins expressed in yeast ormammalian expression systems, e.g., COS-7 cells, may be similar orslightly different in molecular weight and glycosylation pattern thanthe native molecules, depending upon the expression system. Expressionof DNAs encoding the inventive proteins in bacteria such as E. coliprovides non-glycosylated molecules. Functional mutant analogs of RANKprotein having inactivated N-glycosylation sites can be produced byoligonucleotide synthesis and ligation or by site-specific mutagenesistechniques. These analog proteins can be produced in a homogeneous,reduced-carbohydrate form in good yield using yeast expression systems.N-glycosylation sites in eukaryotic proteins are characterized by theamino acid triplet Asn—A₁—Z, where A₁ is any amino acid except Pro, andZ is Ser or Thr. In this sequence, asparagine provides a side chainamino group for covalent attachment of carbohydrate. Such a site can beeliminated by substituting another amino acid for Asn or for residue Z,deleting Asn or Z, or inserting a non-Z amino acid between A₁ and Z, oran amino acid other than Asn between Asn and A₁.

[0037] RANK protein derivatives may also be obtained by mutations of thenative RANK or subunits thereof. A RANK mutated protein, as referred toherein, is a polypeptide homologous to a native RANK protein,respectively, but which has an amino acid sequence different from thenative protein because of one or a plurality of deletions, insertions orsubstitutions. The effect of any mutation made in a DNA encoding amutated peptide may be easily determined by analyzing the ability of themutated peptide to bind its counterstructure in a specific manner.Moreover, activity of RANK analogs, muteins or derivatives can bedetermined by any of the assays described herein (for example,inhibition of the ability of RANK to activate transcription).

[0038] Analogs of the inventive proteins may be constructed by, forexample, making various substitutions of residues or sequences ordeleting terminal or internal residues or sequences not needed forbiological activity. For example, cysteine residues can be deleted orreplaced with other amino acids to prevent formation of incorrectintramolecular disulfide bridges upon renaturation. Other approaches tomutagenesis involve modification of adjacent dibasic amino acid residuesto enhance expression in yeast systems in which KEX2 protease activityis present.

[0039] When a deletion or insertion strategy is adopted, the potentialeffect of the deletion or insertion on biological activity should beconsidered. Subunits of the inventive proteins may be constructed bydeleting terminal or internal residues or sequences. Soluble forms ofRANK can be readily prepared and tested for their ability to inhibitRANK-induced NF-κB activation. Polypeptides corresponding to thecytoplasmic regions, and fragments thereof (for example, a death domain)can be prepared by similar techniques. Additional guidance as to thetypes of mutations that can be made is provided by a comparison of thesequence of RANK to proteins that have similar structures, as well as byperforming structural analysis of the inventive RANK proteins.

[0040] Generally, substitutions should be made conservatively; i.e., themost preferred substitute amino acids are those which do not affect thebiological activity of RANK (i.e., ability of the inventive proteins tobind antibodies to the corresponding native protein in substantiallyequivalent a manner, the ability to bind the counterstructure insubstantially the same manner as the native protein, the ability totransduce a RANK signal, or ability to induce NF-κB activation uponoverexpression in transient transfection systems, for example). Examplesof conservative substitutions include substitution of amino acidsoutside of the binding domain(s) (either ligand/receptor or antibodybinding areas for the extracellular domain, or regions that interactwith other, intracellular proteins for the cytoplasmic domain), andsubstitution of amino acids that do not alter the secondary and/ortertiary structure of the native protein. Additional examples includesubstituting one aliphatic residue for another, such as Ile, Val, Leu,or Ala for one another, or substitutions of one polar residue foranother, such as between Lys and Arg; Glu and Asp; or Gln and Asn. Othersuch conservative substitutions, for example, substitutions of entireregions having similar hydrophobicity characteristics, are well known.

[0041] Mutations in nucleotide sequences constructed for expression ofanalog proteins or fragments thereof must, of course, preserve thereading frame phase of the coding sequences and preferably will notcreate complementary regions that could hybridize to produce secondarymRNA structures such as loops or hairpins which would adversely affecttranslation of the mRNA.

[0042] Not all mutations in the nucleotide sequence which encodes a RANKprotein or fragments thereof will be expressed in the final product, forexample, nucleotide substitutions may be made to enhance expression,primarily to avoid secondary structure loops in the transcribed mRNA(see EPA 75,444A, incorporated herein by reference), or to providecodons that are more readily translated by the selected host, e.g., thewell-known E. coli preference codons for E. coli expression.

[0043] Although a mutation site may be predetermined, it is notnecessary that the nature of the mutation per se be predetermined. Forexample, in order to select for optimum characteristics of mutants,random mutagenesis may be conducted and the expressed mutated proteinsscreened for the desired activity. Mutations can be introduced atparticular loci by synthesizing oligonucleotides containing a mutantsequence, flanked by restriction sites enabling ligation to fragments ofthe native sequence. Following ligation, the resulting reconstructedsequence encodes an analog having the desired amino acid insertion,substitution, or deletion.

[0044] Alternatively, oligonucleotide-directed site-specific mutagenesisprocedures can be employed to provide an altered gene having particularcodons altered according to the substitution, deletion, or insertionrequired. Exemplary methods of making the alterations set forth aboveare disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene37:73, 1985); Craik (BioTechniques, Jan. 12-19 1985); Smith et al.(Genetic Engineering: Principles and Methods, Plenum Press, 1981); andU.S. Pat. Nos. 4,518,584 and 4,737,462 disclose suitable techniques, andare incorporated by reference herein.

[0045] Other embodiments of the inventive proteins include RANKpolypeptides encoded by DNAs capable of hybridizing to the DNA of SEQ IDNO:6 under moderately stringent conditions (prewashing solution of5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0) and hybridization conditions of50° C., 5×SSC, overnight) to the DNA sequences encoding RANK, or morepreferably under stringent conditions (for example, hybridization in6×SSC at 63° C. overnight; washing in 3×SSC at 55° C.), and othersequences which are degenerate to those which encode the RANK. In oneembodiment, RANK polypeptides are at least about 70% identical in aminoacid sequence to the amino acid sequence of native RANK protein as setforth in SEQ ID NO:5. In a preferred embodiment, RANK polypeptides areat least about 80% identical in amino acid sequence to the native formof RANK; most preferred polypeptides are those that are at least about90% identical to native RANK.

[0046] Percent identity may be determined using a computer program, forexample, the GAP computer program described by Devereux et al. (Nucl.Acids Res. 12:387, 1984) and available from the University of WisconsinGenetics Computer Group (UWGCG). For fragments derived from the RANKprotein, the identity is calculated based on that portion of the RANKprotein that is present in the fragment.

[0047] The biological activity of RANK analogs or muteins can bedetermined by testing the ability of the analogs or muteins to inhibitactivation of transcription, for example as described in the Examplesherein. Alternatively, suitable assays, for example, an enzymeimmunoassay or a dot blot, employing an antibody that binds native RANK,or a soluble form of RANKL, can be used to assess the activity of RANKanalogs or muteins, as can assays that employ cells expressing RANKL.Suitable assays also include, for example, signal transduction assaysand methods that evaluate the ability of the cytoplasmic region of RANKto associate with other intracellular proteins (i.e., TRAFs 2 and 3)involved in signal transduction will also be useful to assess theactivity of RANK analogs or muteins. Such methods are well known in theart.

[0048] Fragments of the RANK nucleotide sequences are also useful. Inone embodiment, such fragments comprise at least about 17 consecutivenucleotides, preferably at least about 25 nucleotides, more preferablyat least 30 consecutive nucleotides, of the RANK DNA disclosed herein.DNA and RNA complements of such fragments are provided herein, alongwith both single-stranded and double-stranded forms of the RANK DNA ofSEQ ID NO:5, and those encoding the aforementioned polypeptides. Afragment of RANK DNA generally comprises at least about 17 nucleotides,preferably from about 17 to about 30 nucleotides. Such nucleic acidfragments (for example, a probe corresponding to the extracellulardomain of RANK) are used as a probe or as primers in a polymerase chainreaction (PCR).

[0049] The probes also find use in detecting the presence of RANKnucleic acids in in vitro assays and in such procedures as Northern andSouthern blots. Cell types expressing RANK can be identified as well.Such procedures are well known, and the skilled artisan can choose aprobe of suitable length, depending on the particular intendedapplication. For PCR, 5′ and 3′ primers corresponding to the termini ofa desired RANK DNA sequence are employed to amplify that sequence, usingconventional techniques.

[0050] Other useful fragments of the RANK nucleic acids are antisense orsense oligonucleotides comprising a single-stranded nucleic acidsequence (either RNA or DNA) capable of binding to target RANK mRNA(sense) or RANK DNA (antisense) sequences. The ability to create anantisense or a sense oligonucleotide, based upon a cDNA sequence for agiven protein is described in, for example, Stein and Cohen, Cancer Res.48:2659, 1988 and van der Krol et al., BioTechniques 6:958, 1988.

[0051] Uses of DNAs, Proteins and Analogs

[0052] The RANK DNAs, proteins and analogs described herein will havenumerous uses, including the preparation of pharmaceutical compositions.For example, soluble forms of RANK will be useful as antagonists ofRANK-mediated NF-κB activation, as well as to inhibit transduction of asignal via RANK. RANK compositions (both protein and DNAs) will also beuseful in development of both agonistic and antagonistic antibodies toRANK. The inventive DNAs are useful for the expression of recombinantproteins, and as probes for analysis (either quantitative orqualitative) of the presence or distribution of RANK transcripts.

[0053] The inventive proteins will also be useful in preparing kits thatare used to detect soluble RANK or RANKL, or monitor RANK-relatedactivity, for example, in patient specimens. RANK proteins will alsofind uses in monitoring RANK-related activity in other samples orcompositions, as is necessary when screening for antagonists or mimeticsof this activity (for example, peptides or small molecules that inhibitor mimic, respectively, the interaction). A variety of assay formats areuseful in such kits, including (but not limited to) ELISA, dot blot,solid phase binding assays (such as those using a biosensor), rapidformat assays and bioassays.

[0054] The purified RANK according to the invention will facilitate thediscovery of inhibitors of RANK, and thus, inhibitors of an inflammatoryresponse (via inhibition of NF-κB activation). The use of a purifiedRANK polypeptide in the screening for potential inhibitors is importantand can virtually eliminate the possibility of interfering reactionswith contaminants. Such a screening assay can utilize either theextracellular domain of RANK, the intracellular domain, or a fragment ofeither of these polypeptides. Detecting the inhibiting activity of amolecule would typically involve use of a soluble form of RANK derivedfrom the extracellular domain in a screening assay to detect moleculescapable of binding RANK and inhibiting binding of, for example, anagonistic antibody or RANKL, or using a polypeptide derived from theintracellular domain in an assay to detect inhibition of the interactionof RANK and other, intracellular proteins involved in signaltransduction.

[0055] Moreover, in vitro systems can be used to ascertain the abilityof molecules to antagonize or agonize RANK activity. Included in suchmethods are uses of RANK chimeras, for example, a chimera of the RANKintracellular domain and an extracellular domain derived from a proteinhaving a known ligand. The effects on signal transduction of variousmolecule can then be monitored by utilizing the known ligand totransduce a signal.

[0056] In addition, RANK polypeptides can also be used forstructure-based design of RANK-inhibitors. Such structure-based designis also known as “rational drug design.” The RANK polypeptides can bethree-dimensionally analyzed by, for example, X-ray crystallography,nuclear magnetic resonance or homology modeling, all of which arewell-known methods. The use of RANK structural information in molecularmodeling software systems to assist in inhibitor design is alsoencompassed by the invention. Such computer-assisted modeling and drugdesign may utilize information such as chemical conformational analysis,electrostatic potential of the molecules, protein folding, etc. Aparticular method of the invention comprises analyzing the threedimensional structure of RANK for likely binding sites of substrates,synthesizing a new molecule that incorporates a predictive reactivesite, and assaying the new molecule as described above.

[0057] Expression of Recombinant RANK

[0058] The proteins of the present invention are preferably produced byrecombinant DNA methods by inserting a DNA sequence encoding RANKprotein or an analog thereof into a recombinant expression vector andexpressing the DNA sequence in a recombinant expression system underconditions promoting expression. DNA sequences encoding the proteinsprovided by this invention can be assembled from cDNA fragments andshort oligonucleotide linkers, or from a series of oligonucleotides, toprovide a synthetic gene which is capable of being inserted in arecombinant expression vector and expressed in a recombinanttranscriptional unit.

[0059] Recombinant expression vectors include synthetic or cDNA-derivedDNA fragments encoding RANK, or homologs, muteins or bioequivalentanalogs thereof, operably linked to suitable transcriptional ortranslational regulatory elements derived from mammalian, microbial,viral or insect genes. Such regulatory elements include atranscriptional promoter, an optional operator sequence to controltranscription, a sequence encoding suitable mRNA ribosomal bindingsites, and sequences which control the termination of transcription andtranslation, as described in detail below. The ability to replicate in ahost, usually conferred by an origin of replication, and a selectiongene to facilitate recognition of transformants may additionally beincorporated.

[0060] DNA regions are operably linked when they are functionallyrelated to each other. For example, DNA for a signal peptide (secretoryleader) is operably linked to DNA for a polypeptide if it is expressedas a precursor which participates in the secretion of the polypeptide; apromoter is operably linked to a coding sequence if it controls thetranscription of the sequence; or a ribosome binding site is operablylinked to a coding sequence if it is positioned so as to permittranslation. Generally, operably linked means contiguous and, in thecase of secretory leaders, contiguous and in reading frame. DNAsequences encoding RANK, or homologs or analogs thereof which are to beexpressed in a microorganism will preferably contain no introns thatcould prematurely terminate transcription of DNA into mRNA.

[0061] Useful expression vectors for bacterial use can comprise aselectable marker and bacterial origin of replication derived fromcommercially available plasmids comprising genetic elements of the wellknown cloning vector pBR322 (ATCC 37017). Such commercial vectorsinclude, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala,Sweden) and pGEM1 (Promega Biotec, Madison, Wis., USA). These pBR322“backbone” sections are combined with an appropriate promoter and thestructural sequence to be expressed. E. coli is typically transformedusing derivatives of pBR322, a plasmid derived from an E. coli species(Bolivar et al., Gene 2:95, 1977). pBR322 contains genes for ampicillinand tetracycline resistance and thus provides simple means foridentifying transformed cells.

[0062] Promoters commonly used in recombinant microbial expressionvectors include the β-lactamase (penicillinase) and lactose promotersystem (Chang et al., Nature 275:615, 1978; and Goeddel et al., Nature281:544, 1979), the tryptophan (trp) promoter system (Goeddel et al.,Nucl. Acids Res. 8:4057, 1980; and EPA 36,776) and tac promoter(Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, p. 412, 1982). A particularly useful bacterial expressionsystem employs the phage λ P_(L) promoter and cI857ts thermolabilerepressor. Plasmid vectors available from the American Type CultureCollection which incorporate derivatives of the λ P_(L) promoter includeplasmid pHUB2, resident in E. coli strain JMB9 (ATCC 37092) and pPLc28,resident in E coli RR1 (ATCC 53082).

[0063] Suitable promoter sequences in yeast vectors include thepromoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman etal., J. Biol. Chem. 255:2073, 1980) or other glycolytic enzymes (Hess etal., J. Adv. Enzyme Reg. 7:149, 1968; and Holland et al., Biochem.17:4900, 1978), such as enolase, glyceraldehyde-3-phosphatedehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase. Suitable vectors and promoters for use in yeast expressionare further described in R. Hitzeman et al., EPA 73,657.

[0064] Preferred yeast vectors can be assembled using DNA sequences frompBR322 for selection and replication in E. coli (Amp^(r) gene and originof replication) and yeast DNA sequences including a glucose-repressibleADH2 promoter and α-factor secretion leader. The ADH2 promoter has beendescribed by Russell et al. (J. Biol. Chem. 258:2674, 1982) and Beier etal. (Nature 300:724, 1982). The yeast α-factor leader, which directssecretion of heterologous proteins, can be inserted between the promoterand the structural gene to be expressed. See, e.g., Kurjan et al., Cell30:933, 1982; and Bitter et al., Proc. Natl. Acad. Sci. USA 81:5330,1984. The leader sequence may be modified to contain, near its 3′ end,one or more useful restriction sites to facilitate fusion of the leadersequence to foreign genes.

[0065] The transcriptional and translational control sequences inexpression vectors to be used in transforming vertebrate cells may beprovided by viral sources. For example, commonly used promoters andenhancers are derived from Polyoma, Adenovirus 2, Simian Virus 40(SV40), and human cytomegalovirus. DNA sequences derived from the SV40viral genome, for example, SV40 origin, early and late promoter,enhancer, splice, and polyadenylation sites may be used to provide theother genetic elements required for expression of a heterologous DNAsequence. The early and late promoters are particularly useful becauseboth are obtained easily from the virus as a fragment which alsocontains the SV40 viral origin of replication (Fiers et al., Nature273:113, 1978). Smaller or larger SV40 fragments may also be used,provided the approximately 250 bp sequence extending from the Hind IIIsite toward the BglI site located in the viral origin of replication isincluded. Further, viral genomic promoter, control and/or signalsequences may be utilized, provided such control sequences arecompatible with the host cell chosen. Exemplary vectors can beconstructed as disclosed by Okayama and Berg (Mol. Cell. Biol. 3:280,1983).

[0066] A useful system for stable high level expression of mammalianreceptor cDNAs in C127 murine mammary epithelial cells can beconstructed substantially as described by Cosman et al. (Mol. Immunol.23:935, 1986). A preferred eukaryotic vector for expression of RANK DNAis referred to as pDC406 (McMahan et al., EMBO J. 10:2821, 1991), andincludes regulatory sequences derived from SV40, human immunodeficiencyvirus (HIV), and Epstein-Barr virus (EBV). Other preferred vectorsinclude pDC409 and pDC410, which are derived from pDC406. pDC410 wasderived from pDC406 by substituting the EBV origin of replication withsequences encoding the SV40 large T antigen. pDC409 differs from pDC406in that a Bgl II restriction site outside of the multiple cloning sitehas been deleted, making the Bgl II site within the multiple cloningsite unique.

[0067] A useful cell line that allows for episomal replication ofexpression vectors, such as pDC406 and pDC409, which contain the EBVorigin of replication, is CV-1/EBNA (ATCC CRL 10478). The CV-1/EBNA cellline was derived by transfection of the CV-1 cell line with a geneencoding Epstein-Barr virus nuclear antigen-1 (EBNA-1) andconstitutively express EBNA-1 driven from human CMV immediate-earlyenhancer/promoter.

[0068] Host Cells

[0069] Transformed host cells are cells which have been transformed ortransfected with expression vectors constructed using recombinant DNAtechniques and which contain sequences encoding the proteins of thepresent invention. Transformed host cells may express the desiredprotein (RANK, or homologs or analogs thereof), but host cellstransformed for purposes of cloning or amplifying the inventive DNA donot need to express the protein. Expressed proteins will preferably besecreted into the culture supernatant, depending on the DNA selected,but may be deposited in the cell membrane.

[0070] Suitable host cells for expression of proteins includeprokaryotes, yeast or higher eukaryotic cells under the control ofappropriate promoters. Prokaryotes include gram negative or grampositive organisms, for example E. coli or Bacillus spp. Highereukaryotic cells include established cell lines of mammalian origin asdescribed below. Cell-free translation systems could also be employed toproduce proteins using RNAs derived from the DNA constructs disclosedherein. Appropriate cloning and expression vectors for use withbacterial, fungal, yeast, and mammalian cellular hosts are described byPouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, N.Y.,1985), the relevant disclosure of which is hereby incorporated byreference.

[0071] Prokaryotic expression hosts may be used for expression of RANK,or homologs or analogs thereof that do not require extensive proteolyticand disulfide processing. Prokaryotic expression vectors generallycomprise one or more phenotypic selectable markers, for example a geneencoding proteins conferring antibiotic resistance or supplying anautotrophic requirement, and an origin of replication recognized by thehost to ensure amplification within the host. Suitable prokaryotic hostsfor transformation include E. coli, Bacillus subtilis, Salmonellatyphimurium, and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

[0072] Recombinant RANK may also be expressed in yeast hosts, preferablyfrom the Saccharomyces species, such as S. cerevisiae. Yeast of othergenera, such as Pichia or Kluyveromyces may also be employed. Yeastvectors will generally contain an origin of replication from the 2μyeast plasmid or an autonomously replicating sequence (ARS), promoter,DNA encoding the protein, sequences for polyadenylation andtranscription termination and a selection gene. Preferably, yeastvectors will include an origin of replication and selectable markerpermitting transformation of both yeast and E. coli, e.g., theampicillin resistance gene of E. coli and S. cerevisiae trp1 gene, whichprovides a selection marker for a mutant strain of yeast lacking theability to grow in tryptophan, and a promoter derived from a highlyexpressed yeast gene to induce transcription of a structural sequencedownstream. The presence of the trp1 lesion in the yeast host cellgenome then provides an effective environment for detectingtransformation by growth in the absence of tryptophan.

[0073] Suitable yeast transformation protocols are known to those ofskill in the art; an exemplary technique is described by Hinnen et al.,Proc. Natl. Acad. Sci. USA 75:1929, 1978, selecting for Trp⁺transformants in a selective medium consisting of 0.67% yeast nitrogenbase, 0.5% casamino acids, 2% glucose, 10 μg/ml adenine and 20 μg/mluracil. Host strains transformed by vectors comprising the ADH2 promotermay be grown for expression in a rich medium consisting of 1% yeastextract, 2% peptone, and 1% glucose supplemented with 80 μg/ml adenineand 80 μg/ml uracil. Derepression of the ADH2 promoter occurs uponexhaustion of medium glucose. Crude yeast supernatants are harvested byfiltration and held at 4° C. prior to further purification.

[0074] Various mammalian or insect cell culture systems can be employedto express recombinant protein. Baculovirus systems for production ofheterologous proteins in insect cells are reviewed by Luckow andSummers, Bio/lTechnology 6:47 (1988). Examples of suitable mammalianhost cell lines include the COS-7 lines of monkey kidney cells,described by Gluzman (Cell 23:175, 1981), and other cell lines capableof expressing an appropriate vector including, for example, CV-1/EBNA(ATCC CRL 10478), L cells, C127, 3T3, Chinese hamster ovary (CHO), HeLaand BHK cell lines. Mammalian expression vectors may comprisenontranscribed elements such as an origin of replication, a suitablepromoter and enhancer linked to the gene to be expressed, and other 5′or 3′ flanking nontranscribed sequences, and 5′ or 3′ nontranslatedsequences, such as necessary ribosome binding sites, a polyadenylationsite, splice donor and acceptor sites, and transcriptional terminationsequences.

[0075] Purification of Recombinant RANK

[0076] Purified RANK, and homologs or analogs thereof are prepared byculturing suitable host/vector systems to express the recombinanttranslation products of the DNAs of the present invention, which arethen purified from culture media or cell extracts. For example,supernatants from systems which secrete recombinant protein into culturemedia can be first concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pelliconultrafiltration unit.

[0077] Following the concentration step, the concentrate can be appliedto a suitable purification matrix. For example, a suitable affinitymatrix can comprise a counter structure protein or lectin or antibodymolecule bound to a suitable support. Alternatively, an anion exchangeresin can be employed, for example, a matrix or substrate having pendantdiethylaminoethyl (DEAE) groups. The matrices can be acrylamide,agarose, dextran, cellulose or other types commonly employed in proteinpurification. Alternatively, a cation exchange step can be employed.Suitable cation exchangers include various insoluble matrices comprisingsulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred.Gel filtration chromatography also provides a means of purifying theinventive proteins.

[0078] Affinity chromatography is a particularly preferred method ofpurifying RANK and homologs thereof. For example, a RANK expressed as afusion protein comprising an immunoglobulin Fc region can be purifiedusing Protein A or Protein G affinity chromatography. Moreover, a RANKprotein comprising an oligomerizing zipper domain may be purified on aresin comprising an antibody specific to the oligomerizing zipperdomain. Monoclonal antibodies against the RANK protein may also beuseful in affinity chromatography purification, by utilizing methodsthat are well-known in the art. A ligand may also be used to prepare anaffinity matrix for affinity purification of RANK.

[0079] Finally, one or more reversed-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify a RANK composition. Some or all of theforegoing purification steps, in various combinations, can also beemployed to provide a homogeneous recombinant protein.

[0080] Recombinant protein produced in bacterial culture is usuallyisolated by initial extraction from cell pellets, followed by one ormore concentration, salting-out, aqueous ion exchange or size exclusionchromatography steps. Finally, high performance liquid chromatography(HPLC) can be employed for final purification steps. Microbial cellsemployed in expression of recombinant protein can be disrupted by anyconvenient method, including freeze-thaw cycling, sonication, mechanicaldisruption, or use of cell lysing agents.

[0081] Fermentation of yeast which express the inventive protein as asecreted protein greatly simplifies purification. Secreted recombinantprotein resulting from a large-scale fermentation can be purified bymethods analogous to those disclosed by Urdal et al. (J. Chromatog.296:171, 1984). This reference describes two sequential, reversed-phaseHPLC steps for purification of recombinant human GM-CSF on a preparativeHPLC column.

[0082] Protein synthesized in recombinant culture is characterized bythe presence of cell components, including proteins, in amounts and of acharacter which depend upon the purification steps taken to recover theinventive protein from the culture. These components ordinarily will beof yeast, prokaryotic or non-human higher eukaryotic origin andpreferably are present in innocuous contaminant quantities, on the orderof less than about 1 percent by weight. Further, recombinant cellculture enables the production of the inventive proteins free of otherproteins which may be normally associated with the proteins as they arefound in nature in the species of origin.

[0083] Uses and Administration of RANK Compositions

[0084] The present invention provides methods of using therapeuticcompositions comprising an effective amount of a protein and a suitablediluent and carrier, and methods for regulating an immune orinflammatory response. The use of RANK in conjunction with solublecytokine receptors or cytokines, or other immunoregulatory molecules isalso contemplated.

[0085] For therapeutic use, purified protein is administered to apatient, preferably a human, for treatment in a manner appropriate tothe indication. Thus, for example, RANK protein compositionsadministered to regulate immune function can be given by bolusinjection, continuous infusion, sustained release from implants, orother suitable technique. Typically, a therapeutic agent will beadministered in the form of a composition comprising purified RANK, inconjunction with physiologically acceptable carriers, excipients ordiluents. Such carriers will be nontoxic to recipients at the dosagesand concentrations employed.

[0086] Ordinarily, the preparation of such protein compositions entailscombining the inventive protein with buffers, antioxidants such asascorbic acid, low molecular weight (less than about 10 residues)polypeptides, proteins, amino acids, carbohydrates including glucose,sucrose or dextrins, chelating agents such as EDTA, glutathione andother stabilizers and excipients. Neutral buffered saline or salinemixed with conspecific serum albumin are exemplary appropriate diluents.Preferably, product is formulated as a lyophilizate using appropriateexcipient solutions (e.g., sucrose) as diluents. Appropriate dosages canbe determined in trials. The amount and frequency of administration willdepend, of course, on such factors as the nature and severity of theindication being treated, the desired response, the condition of thepatient, and so forth.

[0087] Soluble forms of RANK and other RANK antagonists such asantagonistic monoclonal antibodies can be administered for the purposeof inhibiting RANK-induced induction of NF-κB activity. NF-κB is atranscription factor that is utilized extensively by cells of the immunesystem, and plays a role in the inflammatory response. Thus, inhibitorsof RANK signalling will be useful in treating conditions in whichsignalling through RANK has given rise to negative consequences, forexample, toxic or septic shock, or graft-versus-host reactions. They mayalso be useful in interfering with the role of NF-κB in cellulartransformation. Tumor cells are more responsive to radiation when theirNF-κB is blocked; thus, soluble RANK (or other antagonists of RANKsignalling) will be useful as an adjunct therapy for diseasecharacterized by neoplastic cells that express RANK.

[0088] The following examples are offered by way of illustration, andnot by way of limitation. Those skilled in the art will recognize thatvariations of the invention embodied in the examples can be made,especially in light of the teachings of the various references citedherein, the disclosures of which are incorporated by reference.

EXAMPLE 1

[0089] The example describes the identification and isolation of a DNAencoding a novel member of the TNF receptor superfamily. A partial cDNAinsert with a predicted open reading frame having some similarity toCD40 (a cell-surface antigen present on the surface of both normal andneoplastic human B cells that has been shown to play an important rolein B-cell proliferation and differentiation; Stamenkovic et al., EMBO J.8:1403, 1989), was identified in a database containing sequenceinformation from cDNAs generated from human bone marrow-deriveddendritic cells (DC). The insert was excised from the vector byrestriction endonuclease digestion, gel purified. labeled with ³²P, andused to hybridize to colony blots generated from a DC cDNA librarycontaining larger cDNA inserts using high stringency hybridization andwashing techniques (hybridization in 5×SSC, 50% formamide at 42° C.overnight, washing in 0.5×SSC at 63° C.); other suitable high stringencyconditions are disclosed in Sambrook et al. in Molecular Cloning: ALaboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.; 1989), 9.52-9.55. Initial experiments yielded a clonereferred to as 9D-8A (SEQ ID NO:1); subsequent analysis indicated thatthis clone contained all but the extreme 5′ end of a novel cDNA, withpredicted intron sequence at the extreme 5′ end (nucleotides 1-92 of SEQID NO:1). Additional colony hybridizations were performed, and a secondclone was isolated. The second clone, referred to as 9D-15C (SEQ IDNO:3), contained the 5′ end without intron interruption but not the full3′ end. SEQ ID NO:5 shows the nucleotide and amino acid sequence of apredicted full-length protein based on alignment of the overlappingsequences of SEQ ID NOs:1 and 3.

[0090] The encoded protein was designated RANK, for receptor activatorof NF-κB. The cDNA encodes a predicted Type 1 transmembrane proteinhaving 616 amino acid residues, with a predicted 24 amino acid signalsequence (the computer predicted cleavage site is after Leu24), a 188amino acid extracellular domain, a 21 amino acid transmembrane domain,and a 383 amino acid cytoplasmic tail. The extracellular region of RANKdisplayed significant amino acid homology (38.5% identity, 52.3%similarity) to CD40. A cloning vector (pBluescriptSK−) containing humanRANK sequence, designated pBluescript:huRANK (in E. coli DH10B), wasdeposited with the American Type Culture Collection, Rockville, Md.(ATCC) on Dec. 20, 1996, under terms of the Budapest Treaty, and givenaccession number 98285.

EXAMPLE 2

[0091] This example describes construction of a RANK DNA construct toexpress a RANK/Fc fusion protein. A soluble form of RANK fused to the Fcregion of human IgG₁ was constructed in the mammalian expression vectorpDC409 (U.S. Ser. No. 08/571,579). This expression vector encodes theleader sequence of the Cytomegalovirus (CMV) open reading frame R27080(SEQ ID NO:9), followed by amino acids 33-213 of RANK, followed by amutated form of the constant domain of human IgG₁ that exhibits reducedaffinity for Fc receptors (SEQ ID NO:8; for the fusion protein, the Fcportion of the construct consisted of Arg3 through Lys232). Analternative expression vector encompassing amino acids 1-213 of RANK(using the native leader sequence) followed by the IgG₁ mutein was alsoprepared. Both expression vectors were found to induce high levels ofexpression of the RANK/Fc fusion protein in transfected cells.

[0092] To obtain RANK/Fc protein, a RANK/Fc expression plasmid istransfected into CV-1/EBNA cells, and supernatants are collected forabout one week. The RANK/Fc fusion protein is purified by meanswell-known in the art for purification of Fc fusion proteins, forexample, by protein A sepharose column chromatography according tomanufacturer's recommendations (i.e., Pharmacia, Uppsala, Sweden).SDS-polyacrylamide gel electrophoresis analysis indicted that thepurified RANK/Fc protein migrated with a molecular weight of ˜55 kDa inthe presence of a reducing agent, and at a molecular weight of ˜110 kDain the absence of a reducing agent.

[0093] N-terminal amino acid sequencing of the purified protein madeusing the CMV R27080 leader showed 60% cleavage after Ala20, 20%cleavage after Pro22 and 20% cleavage after Arg28 (which is the Furincleavage site; amino acid residues are relative to SEQ ID NO:9);N-terminal amino acid analysis of the fusion protein expressed with thenative leader showed cleavage predominantly after Gln25 (80% after Gln25and 20% after Arg23; amino acid residues are relative to SEQ ID NO:6,full-length RANK). Both fusion proteins were able to bind a ligand forRANK is a specific manner (i.e., they bound to the surface of variouscell lines such as a murine thymoma cell line, EL4), indicating that thepresence of additional amino acids at the N-terminus of RANK does notinterfere with its ability to bind RANKL. Moreover, the constructcomprising the CMV leader encoded RANK beginning at amino acid 33; thus,a RANK peptide having an N-terminus at an amino acid between Arg23 andPro33, inclusive, is expected to be able to bind a ligand for RANK in aspecific manner.

[0094] Other members of the TNF receptor superfamily have a region ofamino acids between the transmembrane domain and the ligand bindingdomain that is referred to as a ‘spacer’ region, which is not necessaryfor ligand binding. In RANK, the amino acids between 196 and 213 arepredicted to form such a spacer region. Accordingly, a soluble form ofRANK that terminates with an amino acid in this region is expected toretain the ability to bind a ligand for RANK in a specific manner.Preferred C-terminal amino acids for soluble RANK peptides are selectedfrom the group consisting of amino acids 213 and 196 of SEQ ID NO:6,although other amino acids in the spacer region may be utilized as aC-terminus.

EXAMPLE 3

[0095] This example illustrates the preparation of monoclonal antibodiesagainst RANK. Preparations of purified recombinant RANK, for example, ortransfected cells expressing high levels of RANK, are employed togenerate monoclonal antibodies against RANK using conventionaltechniques, such as those disclosed in U.S. Pat. No. 4,411,993. DNAencoding RANK can also be used as an immunogen, for example, as reviewedby Pardoll and Beckerleg in Immunity 3:165, 1995. Such antibodies arelikely to be useful in interfering with RANK-induced signaling(antagonistic or blocking antibodies) or in inducing a signal bycross-linking RANK (agonistic antibodies), as components of diagnosticor research assays for RANK or RANK activity, or in affinitypurification of RANK.

[0096] To immunize rodents, RANK immunogen is emulsified in an adjuvant(such as complete or incomplete Freund's adjuvant, alum, or anotheradjuvant, such as Ribi adjuvant R700 (Ribi, Hamilton, Mont.), andinjected in amounts ranging from 10-100 μg subcutaneously into aselected rodent, for example, BALB/c mice or Lewis rats. DNA may begiven intradermally (Raz et al., Proc. Natl. Acad. Sci. USA 91:9519,1994) or intamuscularly (Wang et al., Proc. Natl. Acad. Sci. USA90:4156, 1993); saline has been found to be a suitable diluent forDNA-based antigens. Ten days to three weeks days later, the immunizedanimals are boosted with additional immunogen and periodically boostedthereafter on a weekly, biweekly or every third week immunizationschedule.

[0097] Serum samples are periodically taken by retro-orbital bleeding ortail-tip excision for testing by dot-blot assay (antibody sandwich),ELISA (enzyme-linked immunosorbent assay), immunoprecipitation, or othersuitable assays, including FACS analysis. Following detection of anappropriate antibody titer, positive animals are given an intravenousinjection of antigen in saline. Three to four days later, the animalsare sacrificed, splenocytes harvested, and fused to a murine myelomacell line (e.g., NS1 or preferably Ag 8.653 [ATCC CRL 1580]). Hybridomacell lines generated by this procedure are plated in multiple microtiterplates in a selective medium (for example, one containing hypoxanthine,aminopterin, and thyrnidine, or HAT) to inhibit proliferation ofnon-fused cells, myeloma-myeloma hybrids, and splenocyte-splenocytehybrids.

[0098] Hybridoma clones thus generated can be screened by ELISA forreactivity with RANK, for example, by adaptations of the techniquesdisclosed by Engvall et al., Immunochem. 8:871 (1971) and in U.S. Pat.No. 4,703,004. A preferred screening technique is the antibody capturetechnique described by Beckman et al., J. immunol. 144:4212 (1990).Positive clones are then injected into the peritoneal cavities ofsyngeneic rodents to produce ascites containing high concentrations (>1mg/ml) of anti-RANK monoclonal antibody. The resulting monoclonalantibody can be purified by ammonium sulfate precipitation followed bygel exclusion chromatography. Alternatively, affinity chromatographybased upon binding of antibody to protein A or protein G can also beused, as can affinity chromatography based upon binding to RANK protein.

[0099] Monoclonal antibodies were generated using RANK/Fc fusion proteinas the immunogen. These reagents were screened to confirm reactivityagainst the RANK protein. Using the methods described herein to monitorthe activity of the mAbs, both blocking (i.e., antibodies that bind RANKand inhibit binding of a ligand to RANK) and non-blocking (i.e.,antibodies that bind RANK and do not inhibit ligand binding) wereisolated.

EXAMPLE 4

[0100] This example illustrates the induction of NF-κB activity by RANKin 293/EBNA cells (cell line was derived by transfection of the 293 cellline with a gene encoding Epstein-Barr virus nuclear antigen-1 (EBNA-1)that constitutively express EBNA-1 driven from human CMV immediate-earlyenhancer/promoter). Activation of NF-κB activity was measured in293/EBNA cells essentially as described by Yao et al. (Immunity 3:811,1995). Nuclear extracts were prepared and analyzed for NF-κB activity bya gel retardation assay using a 25 base pair oligonucleotide spanningthe NF-κB binding sites. Two million cells were seeded into 10 cm dishestwo days prior to DNA transfection and cultured in DMEM-F12 mediacontaining 2.5% FBS (fetal bovine serum). DNA transfections wereperformed as described herein for the IL-8 promoter/reporter assays.

[0101] Nuclear extracts were prepared by solubilization of isolatednuclei with 400 mM NaCl (Yao et al., supra). Oligonucleotides containingan NF-κB binding site were annealed and endlabeled with ³²P using T4 DNApolynucleotide kinase. Mobility shift reactions contained 10 μg ofnuclear extract, 4 μg of poly(dI-dC) and 15,000 cpm labeleddouble-stranded oligonucleotide and incubated at room temperature for 20minutes. Resulting protein-DNA complexes were resolved on a 6% nativepolyacrylamide gel in 0.25×Tris-borate-EDTA buffer.

[0102] Overexpression of RANK resulted in induction of NF-κB activity asshown by an appropriate shift in the mobility of the radioactive probeon the gel. Similar results were observed when RANK was triggered by aligand that binds RANK and transduces a signal to cells expressing thereceptor (i.e., by co-transfecting cells with human RANK and murineRANKL DNA; see Example 7 below), and would be expected to occur whentriggering is done with agonistic antibodies.

EXAMPLE 5

[0103] This example describes a gene promoter/reporter system based onthe human Interleukin-8 (IL-8 ) promoter used to analyze the activationof gene transcription in vivo. The induction of human IL-8 genetranscription by the cytokines Interleukin-1 (IL-1) or tumor necrosisfactor-alpha (TNF-α) is known to be dependent upon intact NF-κB andNF-IL-6 transcription factor binding sites. Fusion of thecytokine-responsive IL-8 promoter with a cDNA encoding the murine IL4receptor (mIL-4R) allows measurement of promoter activation by detectionof the heterologous reporter protein (mIL-4R) on the cell surface oftransfected cells.

[0104] Human kidney epithelial cells (293/EBNA) are transfected (via theDEAE/DEXTRAN method) with plasmids encoding: 1). the reporter/promoterconstruct (referred to as pIL-8rep), and 2). the cDNA(s) of interest.DNA concentrations are always kept constant by the addition of emptyvector DNA. The 293/EBNA cells are plated at a density of 2.5×10⁴cells/ml (3 ml/well) in a 6 well plate and incubated for two days priorto transfection. Two days after transfection, the mIL-4 receptor isdetected by a radioimmunoassay (RIA) described below.

[0105] In one such experiment, the 293/EBNA cells were co-transfectedwith DNA encoding RANK and with DNA encoding RANKL (see Example 7below). Co-expression of this receptor and its counterstructure by cellsresults in activation of the signaling process of RANK. For suchco-transfection studies, the DNA concentration/well for the DEAEtransfection were as follows: 40 ng of pIL-8rep [pBluescriptSK- vector(Stratagene)]; 0.4 ng CD40 (DNA encoding CD40, a control receptor; pCDM8vector); 0.4 ng RANK (DNA encoding RANK; pDC409 vector), and either 1-50ng CD40L (DNA encoding the ligand for CD40, which acts as a positivecontrol when co-transfected with CD40 and as a negative control whenco-transfected with RANK; in pDC304) or RANKL (DNA encoding a ligand forRANK; in pDC406). Similar experiments can be done using soluble RANKL oragonistic antibodies to RANK to trigger cells transfected with RANK.

[0106] For the mIL-4R-specific RIA, a monoclonal antibody reactive withmIL-4R is labeled with ¹²⁵I via a Chloramine T conjugation method; theresulting specific activity is typically 1.5×10¹⁶ cpm/nmol. After 48hours, transfected cells are washed once with media (DMEM/F12 5% FBS).Non-specific binding sites are blocked by the addition of pre-warmedbinding media containing 5% non-fat dry milk and incubation at 37° C./5%CO₂ in a tissue culture incubator for one hour. The blocking media isdecanted and binding buffer containing ¹²⁵I anti-mIL-4R (clone M1; ratIgG1) is added to the cells and incubated with rocking at roomtemperature for 1 hour. After incubation of the cells with theradio-labled antibody, cells are washed extensively with binding buffer(2×) and twice with phosphate-buffered saline (PBS). Cells are lysed in1 ml of 0.5M NaOH, and total radioactivity is measured with a gammacounter.

[0107] Using this assay, 293/EBNA co-transfected with DNAs encoding RANKdemonstrated transcriptional activation, as shown by detection of muIL4Ron the cell surface. Overexpression of RANK resulted in transcription ofmuIL4R, as did triggering of the RANK by RANKL. Similar results areobserved when RANK is triggered by agonistic antibodies.

EXAMPLE 6

[0108] This example illustrates the association of RANK with TRAFproteins. Interaction of RANK with cytoplasmic TRAF proteins wasdemonstrated by co-immunoprecipitation assays essentially as describedby Hsu et al. (Cell 84:299; 1996). Briefly, 293/EBNA cells wereco-transfected with plasmids that direct the synthesis of RANK andepitope-tagged (FLAG®; SEQ ID NO:7) TRAF2 or TRAF3. Two days aftertransfection, surface proteins were labeled with biotin-ester, and cellswere lysed in a buffer containing 0.5% NP-40. RANK and proteinsassociated with this receptor were immunoprecipitated with anti-RANK,washed extensively, resolved by electrophoretic separation on a 6-10%SDS polyacrylamide gel and electrophoretically transferred to anitrocellulose membrane for Western blotting. The association of TRAF2and TRAF3 proteins with RANK was visualized by probing the membrane withan antibody that specifically recognizes the FLAG® epitope. TRAFs 2 and3 did not immunopreciptitate with anti-RANK in the absence of RANKexpression.

EXAMPLE 7

[0109] This example describes isolation of a ligand for RANK, referredto as RANKL, by direct expression cloning. The ligand was clonedessentially as described in U.S. Ser. No. 08/249,189, filed May 24, 1994(the relevant disclosure of which is incorporated by reference herein),for CD40L. Briefly, a library was prepared from a clone of a mousethymoma cell line EL-4 (ATCC TIB 39), called EL-40.5, derived by sortingfive times with biotinylated CD40/Fc fusion protein in a FACS(fluorescence activated cell sorter). The cDNA library was made usingstandard methodology; the plasmid DNA was isolated and transfected intosub-confluent CV1-EBNA cells using a DEAE-dextran method. Transfectantswere screened by slide autoradiography for expression of RANKL using atwo-step binding method with RANK/Fc fusion protein as prepared inExample 2 followed by radioiodinated goat anti-human IgG antibody.

[0110] A clone encoding a protein that specifically bound RANK wasisolated and sequenced; the clone was referred to as 11H. An expressionvector containing murine RANKL sequence, designated pDC406:muRANK-L (inE. coli DH10B), was deposited with the American Type Culture Collection,Rockville, Md. (ATCC) on Dec. 20, 1996, under terms of the BudapestTreaty, and given accession number 98284. The nucleotide sequence andpredicted amino acid sequence of this clone are illustrated in SEQ IDNO:10. This clone did not contain an initiator methionine; additional,full-length clones were obtained from a 7B9 library (preparedsubstantially as described in U.S. Pat. No. 5,599,905, issued Feb. 4,1997); the 5′ region was found to be identical to that of human RANKL asshown in SEQ ID NO:12, amino acids 1 through 22, except for substitutionof a Gly for a Thr at residue 9.

[0111] This ligand is useful for assessing the ability of RANK to bindRANKL by a number of different assays. For example, transfected cellsexpressing RANKL can be used in a FACS assay (or similar assay) toevaluate the ability of soluble RANK to bind RANKL. Moreover, solubleforms of RANKL can be prepared and used in assays that are known in theart (i.e., ELISA or BIAcore assays essentially as described in U.S. Ser.No. 08/249,189, filed May 24, 1994). RANKL is also useful in affinitypurification of RANK, and as a reagent in methods to measure the levelsof RANK in a sample. Soluble RANKL is also useful in inducing NF-κBactivation and thus protecting cells that express RANK from apoptosis.

EXAMPLE 8

[0112] This example describes the isolation of a human RANK ligand(RANKL) using a PCR-based technique. Murine RANK ligand-specificoligonucleotide primers were used in PCR reactions using human cellline-derived first strand cDNAs as templates. Primers corresponded tonucleotides 478-497 and to the complement of nucleotides 858-878 ofmurine RANK ligand (SEQ ID NO:10). An amplified band approximately 400bp in length from one reaction using the human epidermoid cell line KB(ATCC CCL-17) was gel purified, and its nucleotide sequence determined;the sequence was 85% identical to the corresponding region of murineRANK ligand, confirming that the fragment was from human RANKL.

[0113] To obtain full-length human RANKL cDNAs, two human RANKL-specificoligonucleotides derived from the KB PCR product nucleotide sequencewere radiolabeled and used as hybridization probes to screen a human PBLcDNA library prepared in lambda gt10 (Stratagene, La Jolla, Calif.),substantially as described in U.S. Pat. No. 5,599,905, issued Feb. 4,1997. Several positive hybridizing plaques were identified and purified,their inserts subcloned into pBluescript SK− (Stratagene, La Jolla,Calif.), and their nucleotide sequence determined. One isolate, PBL3,was found to encode most of the predicted human RANKL, but appeared tobe missing approximately 200 bp of 5′ coding region. A second isolate,PBL5 was found to encode much of the predicted human RANKL, includingthe entire 5′ end and an additional 200 bp of 5′ untranslated sequence.

[0114] The 5′ end of PBL5 and the 3′ end of PBL3 were ligated togetherto form a full length cDNA encoding human RANKL. The nucleotide andpredicted amino acid sequence of the full-length human RANK ligand isshown in SEQ ID NO:12. Human RANK ligand shares 83% nucleotide and 84%amino acid identity with murine RANK ligand. A plasmid vector containinghuman RANKL sequence, designated pBluescript:huRANK-L (in E. coliDH10B), was deposited with the American Type Culture Collection,Rockville, Md. (ATCC) on Mar. 11, 1997 under terms of the BudapestTreaty, and given accession number 98354.

[0115] Murine and human RANKL are Type 2 transmembrane proteins. MurineRANKL contains a predicted 48 amino acid intracellular domain, 21 aminoacid transmembrane domain and 247 amino acid extracellular domain. HumanRANKL contains a predicted 47 amino acid intracellular domain, 21 aminoacid transmembrane domain and 249 amino acid extracellular domain.

EXAMPLE 9

[0116] This example describes the chromosomal mapping of human RANKusing PCR-based mapping strategies. Initial human chromosomalassignments were made using RANK and RANKL-specific PCR primers and aBIOS Somatic Cell Hybrid PCRable DNA kit from BIOS Laboratories (NewHaven, Conn.), following the manufacturer's instructions. RANK mapped tohuman chromosome 18; RANK ligand mapped to human chromosome 13. Moredetailed mapping was performed using a radiation hybrid mapping panelGenebridge 4 Radiation Hybrid Panel (Research Genetics, Huntsville,Ala.; described in Walter, Mass. et al., Nature Genetics 7:22-28, 1994).Data from this analysis was then submitted electronically to the MITRadiation Hybrid Mapper (URL: http://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl) following the instructionscontained therein. This analysis yielded specific genetic marker nameswhich, when submitted electronically to the NCBI Entrez browser (URL:http://www3.ncbi.nlm.nih.gov/htbin-post/Entrez/query?db=c&form=0),yielded the specific map locations. RANK mapped to chromosome 18q22. 1,and RANKL mapped to chromosome 13q14.

EXAMPLE 10

[0117] This example illustrates the preparation of monoclonal antibodiesagainst RANKL. Preparations of purified recombinant RANKL, for example,or transfixed cells expressing high levels of RANKL, are employed togenerate monoclonal antibodies against RANKL using conventionaltechniques, such as those disclosed in U.S. Pat. No. 4,411,993. DNAencoding RANKL can also be used as an immunogen, for example, asreviewed by Pardoll and Beckerleg in Immunity 3:165, 1995. Suchantibodies are likely to be useful in interfering with RANKL signaling(antagonistic or blocking antibodies), as components of diagnostic orresearch assays for RANKL or RANKL activity, or in affinity purificationof RANKL.

[0118] To immunize rodents, RANKL immunogen is emulsified in an adjuvant(such as complete or incomplete Freund's adjuvant, alum, or anotheradjuvant, such as Ribi adjuvant R700 (Ribi, Hamilton, Mont.), andinjected in amounts ranging from 10-100 μg subcutaneously into aselected rodent, for example, BALB/c mice or Lewis rats. DNA may begiven intradermally (Raz et al., Proc. Natl. Acad. Sci. USA 91:9519,1994) or intamuscularly (Wang et al., Proc. Natl. Acad. Sci. USA90:4156, 1993); saline has been found to be a suitable diluent forDNA-based antigens. Ten days to three weeks days later, the immunizedanimals are boosted with additional immunogen and periodically boostedthereafter on a weekly, biweekly or every third week immunizationschedule.

[0119] Serum samples are periodically taken by retro-orbital bleeding ortail-tip excision for testing by dot-blot assay (antibody sandwich),ELISA (enzyme-linked immnunosorbent assay), immunoprecipitation, orother suitable assays, including FACS analysis. Following detection ofan appropriate antibody titer, positive animals are given an intravenousinjection of antigen in saline. Three to four days later, the animalsare sacrificed, splenocytes harvested, and fused to a murine myelomacell line (e.g., NS1 or preferably Ag 8.653 [ATCC CRL 1580]). Hybridomacell lines generated by this procedure are plated in multiple microtiterplates in a selective medium (for example, one containing hypoxanthine,aminopterin, and thymidine, or HAT) to inhibit proliferation ofnon-fused cells, myeloma-myeloma hybrids, and splenocyte-splenocytehybrids.

[0120] Hybridoma clones thus generated can be screened by ELISA forreactivity with RANKL, for example, by adaptations of the techniquesdisclosed by Engvall et al., Immunochem. 8:871 (1971) and in U.S. Pat.No. 4,703,004. A preferred screening technique is the antibody capturetechnique described by Beckman et al., J. Immunol. 144:4212 (1990).Positive clones are then injected into the peritoneal cavities ofsyngeneic rodents to produce ascites containing high concentrations (>1mg/ml) of anti-RANK monoclonal antibody. The resulting monoclonalantibody can be purified by ammonium sulfate precipitation followed bygel exclusion chromatography. Alternatively, affinity chromatographybased upon binding of antibody to protein A or protein G can also beused, as can affinity chromatography based upon binding to RANKLprotein. Using the methods described herein to monitor the activity ofthe mAbs, both blocking (i.e., antibodies that bind RANKL and inhibitbinding to RANK) and non-blocking (i.e., antibodies that bind RANKL anddo not inhibit binding) are isolated.

EXAMPLE 11

[0121] This example demonstrates that RANK expression can beup-regulated. Human peripheral blood T cells were purified by flowcytometry sorting or by negative selection using antibody coated beads,and activated with anti-CD3 (OKT3, Dako) coated plates orphytohemagglutinin in the presence or absence of various cytokines,including Interleukin-4 (IL-4), Transforming Growth Factor-β(TGF-β) andother commercially available cytokines ( IL1-α, IL-2, IL-3, IL-6, IL-7,IL-8, IL-10, IL-12, IL-15, IFN-₆₅ , TNF-α). Expression of RANK wasevaluated by FACS in a time course experiment for day 2 to day 8, usinga mouse monoclonal antibody mAb144 (prepared as described in Example 3),as shown in the table below. Results are expressed as ‘+’ to ‘++++’referring to the relative increase in intensity of staining withanti-RANK. Double labeling experiments using both anti-RANK and anti-CD8or anti-CD4 antibodies were also performed. TABLE 1 Upregulation of RANKby Cytokines Cytokine (concentration) Results: IL-4 (50 ng/ml) + TGF-β(5 ng/ml) + to ++ IL-4 (50 ng/ml) + TGF-β (5 ng/ml) ++++ IL1-α (10ng/ml) − IL-2 (20 ng/ml) − IL-3 (25 ng/ml) − IL-7 (20 ng/ml) − IL-8 (10ng/ml) − IL-10 (50 ng/ml) − IL-12 (10 ng/ml) − IL-15 (10 ng/ml) − IFN-γ(100 U/ml) − TNF-α (10 ng/ml) −

[0122] Of the cytokines tested, IL-4 and TGF-β increased the level ofRANK expression on both CD8+ cytotoxic and CD4+ helper T cells from day4 to day 8. The combination of IL-4 and TGF-β acted synergistically toupregulate expression of this receptor on activated T cells. Thisparticular combination of cytokines is secreted by suppresser T cells,and is believed to be important in the generation of tolerance (reviewedin Mitchison and Sieper, Z. Rheumatol. 54:141, 1995), implicating theinteraction of RANK in regulation of an immune response towards eithertolerance or induction of an active immune response.

EXAMPLE 12

[0123] This example illustrates the influence of RANK.Fc and hRANKL onactivated T cell growth. The addition of TGFβ to anti-CD3 activatedhuman peripheral blood T lymphocytes induces proliferation arrest andultimately death of most lymphocytes within the first few days ofculture. We tested the effect of RANK:RANKL interactions on TGFβ-treatedT cells by adding RANK.Fc or soluble human RANKL to T cell cultures.

[0124] Human peripheral blood T cells (7×10⁵ PBT) were cultured for sixdays on anti-CD3 (OKT3, 5μg/ml) and anti-Flag (M1, 5 μg/ml) coated 24well plates in the presence of TGFβ (1 ng/ml) and IL-4 (10 ng/ml), withor without recombinant FLAG-tagged soluble hRANKL (1 μg/ml) or RANK.Fc(10 μg/ml). Viable T cell recovery was determined by triplicate trypanblue countings.

[0125] The addition of RANK.Fc significantly reduced the number ofviable T cells recovered after six days, whereas soluble RANKL greatlyincreased the recovery of viable T cells (FIG. 1). Thus, endogenous orexogenous RANKL enhances the number of viable T cells generated in thepresence of TGFβ. TGFβ, along with IL-4, has been implicated in immuneresponse regulation when secreted by the T_(H)3/regulatory T cellsubset. These T cells are believed to mediate bystander suppression ofeffector T cells. Accordingly, RANK and its ligand may act in anauto/paracrine fashion to influence T cell tolerance. Moreover, TGFβ isknown to play a role in the evasion of the immune system effected bycertain pathogenic or opportunistic organisms. In addition to playing arole in the development of tolerance, RANK may also play a role inimmune system evasion by pathogens.

EXAMPLE 13

[0126] This example illustrates the influence of the interaction of RANKon CD1a³⁰ dendritic cells (DC). Functionally mature dendritic cells (DC)were generated in vitro from CD34⁺ bone marrow (BM) progenitors.Briefly, human BM cells from normal healthy volunteers were densityfractionated using Ficoll medium and CD34⁺ cells immunoaffinity isolatedusing an anti-CD34 matrix column (Ceprate, CellPro). The CD34⁺ BM cellswere then cultured in human GM-CSF (20 ng/ml), human IL-4 (20 ng/ml),human TNF-α(20 ng/ml), human CHO-derived Flt3L (FL; 100 ng/ml) in SuperMcCoy's medium supplemented with 10% fetal calf serum in a fullyhumidified 37° C. incubator (5% CO₂) for 14 days. CD1a⁺, HLA-DR⁺ DC werethen sorted using a FACStar Plus™, and used for biological evaluation ofRANK.

[0127] On human CD1a⁺ DC derived from CD34⁺ bone marrow cells, only asubset (20-30%) of CD1a⁺ DC expressed RANK at the cell surface asassessed by flow cytometric analysis. However, addition of CD40L to theDC cultures resulted in RANK surface expression on the majority of CD1a⁺DC. CD40L has been shown to activate DC by enhancing in vitro clusterformation, inducing DC morphological changes and upregulating HLA-DR,CD54, CD58, CD80 and CD86 expression

[0128] Addition of RANKL to DC cultures significantly increased thedegree of DC aggregation and cluster formation above control cultures,similar to the effects seen with CD40L (FIG. 2). Sorted human CD1a⁺ DCwere cultured in a cytokine cocktail (GM-CSF, IL-4, TNF-α and FL) (upperleft panel), in cocktail plus CD40L (1 μg/ml) (upper right), in cocktailplus RANKL (1 μg/ml) (lower left), or in cocktail plus heat inactivated(ΔH) RANKL (1 μg/ml) (lower right) in 24-well flat bottomed cultureplates in 1 ml culture media for 48-72 hours and then photographed usingan inversion microscope. An increase in DC aggregation and clusterformation above control cultures was not evident when heat inactivatedRANKL was used, indicating that this effect was dependent onbiologically active protein. However, initial phenotypic analysis ofadhesion molecule expression indicated that RANKL-induced clustering wasnot due to increased levels of CD2, CD11a, CD54 or CD58.

[0129] The addition of RANKL to CD1a⁺ DC enhanced their allo-stimulatorycapacity in a mixed lymphocyte reaction (MLR) by at least 3- to 10-fold,comparable to CD40L-cultured DC (FIG. 3). Allogeneic T cells (1×10⁵)were incubated with varying numbers of irradiated (2000 rad) DC culturedas indicated above for FIG. 2 in 96-well round bottomed culture platesin 0.2 ml culture medium for four days. The cultures were pulsed with0.5 mCi [³H]-thymidine for eight hours and the cells harvested ontoglass fiber sheets for counting on a gas phase β counter. The backgroundcounts for either T cells or DC cultured alone were <100 cpm. Valuesrepresent the mean ±SD of triplicate cultures. Heat inactivated RANKLhad no effect. DC allo-stimulatory activity was not further enhancedwhen RANKL and CD40L were used in combination, possibly due to DCfunctional capacity having reached a maximal level with either cytokinealone. Neither RANKL nor CD40L enhanced the in vitro growth of DC overthe three day culture period. Unlike CD40L, RANKL did not significantlyincrease the levels of HLA-DR expression nor the expression of CD80 orCD86.

[0130] RANKL can enhance DC cluster formation and functional capacitywithout modulating known molecules involved in cell adhesion (CD18,CD54), antigen presentation (HLA-DR) or costimulation (CD86), all ofwhich are regulated by CD40/CD40L signaling. The lack of an effect onthe expression of these molecules suggests that RANKL may regulate DCfunction via an alternate pathway(s) distinct from CD40/CD40L. Giventhat CD40L regulates RANK surface expression on in vitro-generated DCand that CD40L is upregulated on activated T cells during DC-T cellinteractions, RANK and its ligand may form an important part of theactivation cascade that is induced during DC-mediated T cell expansion.Furthermore, culture of DC in RANKL results in decreased levels ofCD1b/c expression, and increased levels of CD83. Both of these moleculesare similarly modulated during DC maturation by CD40L (Caux et al. J.Exp. Med. 180:1263; 1994), indicating that RANKL induces DC maturation.

[0131] Dendritic cells are referred to as “professional” antigenpresenting cells, and have a high capacity for sensitizingMHC-restricted T cells. There is growing interest in using dendriticcells ex vivo as tumor or infectious disease vaccine adjuvants (see, forexample, Romani, et al., J. Exp. Med., 180:83, 1994). Therefore, anagent such as RANKL that induces DC maturation and enhances the abilityof dendritic cells to stimulate an immune response is likely to beuseful in immunotherapy of various diseases.

EXAMPLE 14

[0132] This example describes the isolation of the murine homolog ofRANK, referred to as muRANK. MuRANK was isolated by a combination ofcross-species PCR and colony hybridization. The conservation of Cysresidues in the Cys-rich pseudorepeats of the extracellular domains ofTNFR superfamily member proteins was exploited to design humanRANK-based PCR primers to be used on murine first strand cDNAs fromvarious sources. Both the sense upstream primer and the antisensedownstream primer were designed to have their 3′ ends terminate withinCys residues.

[0133] The upstream sense primer encoded nucleotides 272-295 of SEQ IDNO:5 (region encoding amino acids 79-86); the downstream antisenseprimer encoded the complement of nucleotides 409-427 (region encodingamino acids 124-130). Standard PCR reactions were set up and run, usingthese primers and first strand cDNAs from various murine cell line ortissue sources. Thirty reaction cycles of 94° C. for 30 seconds, 50° C.for 30 seconds, and 72° C. for 20 seconds were run. PCR products wereanlyzed by electrophoresis, and specific bands were seen in severalsamples. The band from one sample was gel purified and DNA sequencingrevealed that the sequence between the primers was approximately 85%identical to the corresponding human RANK nucleotide sequence.

[0134] A plasmid based cDNA library prepared from the murine fetal liverepithelium line FLE18 (one of the cell lines identified as positive inthe PCR screen) was screened for full-length RANK cDNAs using murineRANK-specific oligonucleotide probes derived from the murine RANKsequence determined from sequencing the PCR product. Two cDNAs, oneencoding the 5′ end and one encoding the 3′ end of full-length murineRANK (based on sequence comparison with the full-length human RANK) wererecombined to generate a full-length murine RANK cDNA. The nucleotideand amino acid seqeunce of muRANK are shown in SEQ ID Nos:14 and 15.

[0135] The cDNA encodes a predicted Type 1 transmembrane protein having625 amino acid residues, with a predicted 30 amino acid signal sequence,a 184 amino acid extracellular domain, a 21 amino acid transmembranedomain, and a 390 amino acid cytoplasmic tail. The extracellular regionof muRANK displayed significant amino acid homology (69.7% identity,80.8% similarity) to huRANK. Those of skill in the art will recognizethat the actual cleavage site can be different from that predicted bycomputer; accordingly, the N-terminal of RANK may be from amino acid 25to amino acid 35.

[0136] Other members of the TNF receptor superfamily have a region ofamino acids between the transmembrane domain and the ligand bindingdomain that is referred to as a ‘spacer’ region, which is not necessaryfor ligand binding. In muRANK, the amino acids between 197 and 214 arepredicted to form such a spacer region. Accordingly, a soluble form ofRANK that terminates with an amino acid in this region is expected toretain the ability to bind a ligand for RANK in a specific manner.Preferred C-terminal amino acids for soluble RANK peptides are selectedfrom the group consisting of amino acids 214, and 197 of SEQ ID NO:14,although other amino acids in the spacer region may be utilized as aC-terminus.

EXAMPLE 15

[0137] This example illustrates the preparation of several differentsoluble forms of RANK and RANKL. Standard techniques of restrictionenzyme cutting and ligation, in combination with PCR-based isolation offragments for which no convenient restriction sites existed, were used.When PCR was utilized, PCR products were sequenced to ascertain whetherany mutations had been introduced; no such mutations were found.

[0138] In addition to the huRANK/Fc described in Example 2, anotherRANK/Fc fusion protein was prepared by ligating DNA encoding amino acids1-213 of SEQ ID NO:6, to DNA encoding amino acids 3-232 of the Fc muteindescribed previously (SEQ ID NO:8). A similar construct was prepared formurine RANK, ligating DNA encoding amino acids 1-213 of full-lengthmurine RANK (SEQ ID NO:15) to DNA encoding amino acids 3-232 of the Fcmutein (SEQ ID NO:8).

[0139] A soluble, tagged, poly-His version of huRANKL was prepared byligating DNA encoding the leader peptide from the immunoglobulin kappachain (SEQ ID NO:16) to DNA encoding a short version of the FLAG™ tag(SEQ ID NO:17), followed by codons encoding Gly Ser, then a poly-His tag(SEQ ID NO:18), followed by codons encoding Gly Thr Ser, and DNAencoding amino acids 138-317 of SEQ ID NO:13. A soluble, poly-His taggedversion of murine RANKL was prepared by ligating DNA encoding the CMVleader (SEQ ID NO:9) to codons encoding Arg Thr Ser, followed by DNAencoding poly-His (SEQ ID NO:18) followed by DNA encoding amino acids119-294 of SEQ ID NO:11.

[0140] A soluble, oligomeric form of huRANKL was prepared by ligatingDNA encoding the CMV leader (SEQ ID NO:9) to a codon encoding Aspfollowed by DNA ending a trimer-former “leucine” zipper (SEQ ID NO:19),then by codons encoding Thr Arg Ser followed by amino acids 138-317 ofSEQ ID NO:13.

[0141] These and other constructs are prepared by routineexperimentation. The various DNAs are then inserted into a suitableexpression vector, and expressed. Particularly preferred expressionvectors are those which can be used in mammalian cells. For example,pDC409 and pDC304, described herein, are useful for transientexpression. For stable transfection, the use of CHO cells is preferred;several useful vectors are described in U.S. Ser. No. 08/785,150, nowallowed, for example, one of the 2A5-3 λ-derived expression vectorsdiscussed therein.

EXAMPLE 16

[0142] This example demonstrates that RANKL expression can beup-regulated on murine T cells. Cells were obtained from mesentericlymph nodes of C57BL/6 mice, and activated with anti-CD3 coated plates,Concanavalin A (ConA) or phorbol myristate acetate in combination withionomycin (anti-CD3: 500A2; Immunex Corporation, Seattle Wash.; ConA,PMA, ionomycin, Sigma, St. Louis, Mo.) substantially as describedherein, and cultured from about 2 to 5 days. Expression of RANKL wasevaluated in a three color analysis by FACS, using antibodies to the Tcell markers CD4, CD8 and CD45RB, and RANK/Fc, prepared as describedherein.

[0143] RANKL was not expressed on unstimulated murine T cells. T cellsstimulated with either anti-CD3, ConA, or PMA/ionomycin, showeddifferential expression of RANKL: CD4⁺/CD45RB^(Lo) and CD4⁺/CD45RB^(Hi)cells were positive for RANKL, but CD8+ cells were not. RANKL was notobserved on B cells, similar to results observed with human cells.

EXAMPLE 17

[0144] This example illustrates the effects of murine RANKL on cellproliferation and activation. Various cells or cell lines representativeof cells that play a role in an immune response (murine spleen, thymusand lymphnode) were evaluated by culturing them under conditionspromoting their viability, in the presence or absence of RANKL. RANKLdid not stimulate any of the tested cells to proliferate. One cell line,a macrophage cell line referred to as RAW 264.7 (ATCC accession numberTIB 71) exhibited some signs of activation.

[0145] RAW cells constitutively produce small amounts of TNF-α.Incubation with either human or murine RANKL enhanced production ofTNF-α by these cells in a dose dependent manner. The results were notdue to contamination of RANKL preparations with endotoxin, since boilingRANKL for 10 minutes abrogated TNF-α production, whereas a similartreatment of purified endotoxin (LPS) did not affect the ability of theLPS to stimulate TNF-α production. Despite the fact that RANKL activatedthe macrophage cell line RAW T64.7 for TNF-α production, neither humanRANKL nor murine RANKL stimulated nitric oxide production by thesecells.

EXAMPLE 18

[0146] This example illustrates the effects of murine RANKL on growthand development of the thymus in fetal mice. Pregnant mice were injectedwith 1 mg of RANK/Fc or vehicle control protein (murine serum albumin;MSA) on days 13, 16 and 19 of gestation. After birth, the neonatescontinued to be injected with RANK/Fc intraperitoneally (IP) on a dailybasis, beginning at a dose of 1 μg, and doubling the dose about everyfour days, for a final dosage of 4 μg. Neonates were taken at days 1, 8and 15 post birth, their thymuses and spleens harvested and examined forsize, cellularity and phenotypic composition.

[0147] A slight reduction in thymic size at day 1 was observed in theneonates born to the female injected with RANK/Fc; a similar decrease insize was not observed in the control neonates. At day 8, thymic size andcellularity were reduced by about 50% in the RANK/Fc-treated animals ascompared to MSA treated mice. Phenotypic analysis demonstrated that therelative proportions of different T cell populations in the thymus werethe same in the RANK/Fc mice as the control mice, indicating that thedecreased cellularity was due to a global depression in the number ofthymic T cells as opposed to a decrease in a specific population(s). TheRANK/Fc-treated neonates were not significantly different from thecontrol neonates at day 15 with respect to either size, cellularity orphenotype of thymic cells. No significant differences were observed inspleen size, cellularity or composition at any of the time pointsevaluated. The difference in cellularity on day 8 and not on day 15 maysuggest that RANK/Fc may assert its effect early in thymic development.

EXAMPLE 19

[0148] This example demonstrates that the C-terminal region of thecytoplasmic domain of RANK is important for binding of several differentTRAF proteins. RANK contains at least two recognizable PXQX(X)T motifsthat are likely TRAF docking sites. Accordingly, the importance ofvarious regions of the cytoplasmic domain of RANK for TRAF binding wasevaluated. A RANK/GST fusion protein was prepared substantially asdescribed in Smith and Johnson, Gene 67:31 (1988), and used in thepreparation of various truncations as described below.

[0149] Comparison of the nucleotide sequence of murine and human RANKindicated that there were several conserved regions that could beimportant for TRAF binding. Accordingly, a PCR-based technique wasdeveloped to facilitate preparation of various C-terminal truncationsthat would retain the conserved regions. PCR primers were designed tointroduce a stop codon and restriction enzyme site at selected points,yielding the truncations described in Table 1 below. Sequencingconfirmed that no undesired mutations had been introduced in theconstructs.

[0150] Radio-labeled (³⁵S-Met, Cys) TRAF proteins were prepared by invitro translation using a commercially available reticulocyte lysate kitaccording to manufacturer's instructions (Promega). Truncated GST fusionproteins were purified substantially as described in Smith and Johnson(supra). Briefly, E. coli were transfected with an expression vectorencoding a fusion protein, and induced to express the protein. Thebacteria were lysed, insoluble material removed, and the fusion proteinisolated by precipitation with glutathione-coated beads (Sepahrose 4B,Pharmacia, Uppsala Sweden)

[0151] The beads were washed, and incubated with various radiolabeledTRAF proteins. After incubation and wash steps, the fusion protein/TRAFcomplexes were removed from the beads by boiling in 0.1%SDS+α-mercaptoethanol, and loaded onto 12% SDS gels (Novex). The gelswere subjected to autoradiography, and the presence or absence ofradiolabeled material recorded. The results are shown in Table 2 below.TABLE 2 Binding of Various TRAF Proteins to the Cytoplasmic Domain ofRANK C terminal E206- E206- Truncations: S339 Y421 E206-M476 E206-G544Full length TRAF1 − − − − ++ TRAF2 − − − − ++ TRAF3 − − − − ++ TRAF4 − −− − − TRAF5 − − − − + TRAF6 − + + + ++

[0152] These results indicate that TRAF1, TRAF2, TRAF3, TRAF 5 and TRAF6bind to the most distal portion of the RANK cytoplasmic domain (betweenamino-acid G544 and A616). TRAF6 also has a binding site between S339and Y421. In this experiment, TRAF5 also bound the cytoplasmic domain ofRANK.

1 19 3115 base pairs nucleic acid single linear cDNA NO NO HOMO SAPIENSBONE-MARROW DERIVED DENDRITIC CELLS 9D-8A CDS 93..1868 1 GCTGCTGCTGCTCTGCGCGC TGCTCGCCCG GCTGCAGTTT TATCCAGAAA GAGCTGTGTG 60 GACTCTCTGCCTGACCTCAG TGTTCTTTTC AG GTG GCT TTG CAG ATC GCT CCT 113 Val Ala Leu GlnIle Ala Pro 1 5 CCA TGT ACC AGT GAG AAG CAT TAT GAG CAT CTG GGA CGG TGCTGT AAC 161 Pro Cys Thr Ser Glu Lys His Tyr Glu His Leu Gly Arg Cys CysAsn 10 15 20 AAA TGT GAA CCA GGA AAG TAC ATG TCT TCT AAA TGC ACT ACT ACCTCT 209 Lys Cys Glu Pro Gly Lys Tyr Met Ser Ser Lys Cys Thr Thr Thr Ser25 30 35 GAC AGT GTA TGT CTG CCC TGT GGC CCG GAT GAA TAC TTG GAT AGC TGG257 Asp Ser Val Cys Leu Pro Cys Gly Pro Asp Glu Tyr Leu Asp Ser Trp 4045 50 55 AAT GAA GAA GAT AAA TGC TTG CTG CAT AAA GTT TGT GAT ACA GGC AAG305 Asn Glu Glu Asp Lys Cys Leu Leu His Lys Val Cys Asp Thr Gly Lys 6065 70 GCC CTG GTG GCC GTG GTC GCC GGC AAC AGC ACG ACC CCC CGG CGC TGC353 Ala Leu Val Ala Val Val Ala Gly Asn Ser Thr Thr Pro Arg Arg Cys 7580 85 GCG TGC ACG GCT GGG TAC CAC TGG AGC CAG GAC TGC GAG TGC TGC CGC401 Ala Cys Thr Ala Gly Tyr His Trp Ser Gln Asp Cys Glu Cys Cys Arg 9095 100 CGC AAC ACC GAG TGC GCG CCG GGC CTG GGC GCC CAG CAC CCG TTG CAG449 Arg Asn Thr Glu Cys Ala Pro Gly Leu Gly Ala Gln His Pro Leu Gln 105110 115 CTC AAC AAG GAC ACA GTG TGC AAA CCT TGC CTT GCA GGC TAC TTC TCT497 Leu Asn Lys Asp Thr Val Cys Lys Pro Cys Leu Ala Gly Tyr Phe Ser 120125 130 135 GAT GCC TTT TCC TCC ACG GAC AAA TGC AGA CCC TGG ACC AAC TGTACC 545 Asp Ala Phe Ser Ser Thr Asp Lys Cys Arg Pro Trp Thr Asn Cys Thr140 145 150 TTC CTT GGA AAG AGA GTA GAA CAT CAT GGG ACA GAG AAA TCC GATGCG 593 Phe Leu Gly Lys Arg Val Glu His His Gly Thr Glu Lys Ser Asp Ala155 160 165 GTT TGC AGT TCT TCT CTG CCA GCT AGA AAA CCA CCA AAT GAA CCCCAT 641 Val Cys Ser Ser Ser Leu Pro Ala Arg Lys Pro Pro Asn Glu Pro His170 175 180 GTT TAC TTG CCC GGT TTA ATA ATT CTG CTT CTC TTC GCG TCT GTGGCC 689 Val Tyr Leu Pro Gly Leu Ile Ile Leu Leu Leu Phe Ala Ser Val Ala185 190 195 CTG GTG GCT GCC ATC ATC TTT GGC GTT TGC TAT AGG AAA AAA GGGAAA 737 Leu Val Ala Ala Ile Ile Phe Gly Val Cys Tyr Arg Lys Lys Gly Lys200 205 210 215 GCA CTC ACA GCT AAT TTG TGG CAC TGG ATC AAT GAG GCT TGTGGC CGC 785 Ala Leu Thr Ala Asn Leu Trp His Trp Ile Asn Glu Ala Cys GlyArg 220 225 230 CTA AGT GGA GAT AAG GAG TCC TCA GGT GAC AGT TGT GTC AGTACA CAC 833 Leu Ser Gly Asp Lys Glu Ser Ser Gly Asp Ser Cys Val Ser ThrHis 235 240 245 ACG GCA AAC TTT GGT CAG CAG GGA GCA TGT GAA GGT GTC TTACTG CTG 881 Thr Ala Asn Phe Gly Gln Gln Gly Ala Cys Glu Gly Val Leu LeuLeu 250 255 260 ACT CTG GAG GAG AAG ACA TTT CCA GAA GAT ATG TGC TAC CCAGAT CAA 929 Thr Leu Glu Glu Lys Thr Phe Pro Glu Asp Met Cys Tyr Pro AspGln 265 270 275 GGT GGT GTC TGT CAG GGC ACG TGT GTA GGA GGT GGT CCC TACGCA CAA 977 Gly Gly Val Cys Gln Gly Thr Cys Val Gly Gly Gly Pro Tyr AlaGln 280 285 290 295 GGC GAA GAT GCC AGG ATG CTC TCA TTG GTC AGC AAG ACCGAG ATA GAG 1025 Gly Glu Asp Ala Arg Met Leu Ser Leu Val Ser Lys Thr GluIle Glu 300 305 310 GAA GAC AGC TTC AGA CAG ATG CCC ACA GAA GAT GAA TACATG GAC AGG 1073 Glu Asp Ser Phe Arg Gln Met Pro Thr Glu Asp Glu Tyr MetAsp Arg 315 320 325 CCC TCC CAG CCC ACA GAC CAG TTA CTG TTC CTC ACT GAGCCT GGA AGC 1121 Pro Ser Gln Pro Thr Asp Gln Leu Leu Phe Leu Thr Glu ProGly Ser 330 335 340 AAA TCC ACA CCT CCT TTC TCT GAA CCC CTG GAG GTG GGGGAG AAT GAC 1169 Lys Ser Thr Pro Pro Phe Ser Glu Pro Leu Glu Val Gly GluAsn Asp 345 350 355 AGT TTA AGC CAG TGC TTC ACG GGG ACA CAG AGC ACA GTGGGT TCA GAA 1217 Ser Leu Ser Gln Cys Phe Thr Gly Thr Gln Ser Thr Val GlySer Glu 360 365 370 375 AGC TGC AAC TGC ACT GAG CCC CTG TGC AGG ACT GATTGG ACT CCC ATG 1265 Ser Cys Asn Cys Thr Glu Pro Leu Cys Arg Thr Asp TrpThr Pro Met 380 385 390 TCC TCT GAA AAC TAC TTG CAA AAA GAG GTG GAC AGTGGC CAT TGC CCG 1313 Ser Ser Glu Asn Tyr Leu Gln Lys Glu Val Asp Ser GlyHis Cys Pro 395 400 405 CAC TGG GCA GCC AGC CCC AGC CCC AAC TGG GCA GATGTC TGC ACA GGC 1361 His Trp Ala Ala Ser Pro Ser Pro Asn Trp Ala Asp ValCys Thr Gly 410 415 420 TGC CGG AAC CCT CCT GGG GAG GAC TGT GAA CCC CTCGTG GGT TCC CCA 1409 Cys Arg Asn Pro Pro Gly Glu Asp Cys Glu Pro Leu ValGly Ser Pro 425 430 435 AAA CGT GGA CCC TTG CCC CAG TGC GCC TAT GGC ATGGGC CTT CCC CCT 1457 Lys Arg Gly Pro Leu Pro Gln Cys Ala Tyr Gly Met GlyLeu Pro Pro 440 445 450 455 GAA GAA GAA GCC AGC AGG ACG GAG GCC AGA GACCAG CCC GAG GAT GGG 1505 Glu Glu Glu Ala Ser Arg Thr Glu Ala Arg Asp GlnPro Glu Asp Gly 460 465 470 GCT GAT GGG AGG CTC CCA AGC TCA GCG AGG GCAGGT GCC GGG TCT GGA 1553 Ala Asp Gly Arg Leu Pro Ser Ser Ala Arg Ala GlyAla Gly Ser Gly 475 480 485 AGC TCC CCT GGT GGC CAG TCC CCT GCA TCT GGAAAT GTG ACT GGA AAC 1601 Ser Ser Pro Gly Gly Gln Ser Pro Ala Ser Gly AsnVal Thr Gly Asn 490 495 500 AGT AAC TCC ACG TTC ATC TCC AGC GGG CAG GTGATG AAC TTC AAG GGC 1649 Ser Asn Ser Thr Phe Ile Ser Ser Gly Gln Val MetAsn Phe Lys Gly 505 510 515 GAC ATC ATC GTG GTC TAC GTC AGC CAG ACC TCGCAG GAG GGC GCG GCG 1697 Asp Ile Ile Val Val Tyr Val Ser Gln Thr Ser GlnGlu Gly Ala Ala 520 525 530 535 GCG GCT GCG GAG CCC ATG GGC CGC CCG GTGCAG GAG GAG ACC CTG GCG 1745 Ala Ala Ala Glu Pro Met Gly Arg Pro Val GlnGlu Glu Thr Leu Ala 540 545 550 CGC CGA GAC TCC TTC GCG GGG AAC GGC CCGCGC TTC CCG GAC CCG TGC 1793 Arg Arg Asp Ser Phe Ala Gly Asn Gly Pro ArgPhe Pro Asp Pro Cys 555 560 565 GGC GGC CCC GAG GGG CTG CGG GAG CCG GAGAAG GCC TCG AGG CCG GTG 1841 Gly Gly Pro Glu Gly Leu Arg Glu Pro Glu LysAla Ser Arg Pro Val 570 575 580 CAG GAG CAA GGC GGG GCC AAG GCT TGAGCGCCCCCCA TGGCTGGGAG 1888 Gln Glu Gln Gly Gly Ala Lys Ala 585 590CCCGAAGCTC GGAGCCAGGG CTCGCGAGGG CAGCACCGCA GCCTCTGCCC CAGCCCCGGC 1948CACCCAGGGA TCGATCGGTA CAGTCGAGGA AGACCACCCG GCATTCTCTG CCCACTTTGC 2008CTTCCAGGAA ATGGGCTTTT CAGGAAGTGA ATTGATGAGG ACTGTCCCCA TGCCCACGGA 2068TGCTCAGCAG CCCGCCGCAC TGGGGCAGAT GTCTCCCCTG CCACTCCTCA AACTCGCAGC 2128AGTAATTTGT GGCACTATGA CAGCTATTTT TATGACTATC CTGTTCTGTG GGGGGGGGGT 2188CTATGTTTTC CCCCCATATT TGTATTCCTT TTCATAACTT TTCTTGATAT CTTTCCTCCC 2248TCTTTTTTAA TGTAAAGGTT TTCTCAAAAA TTCTCCTAAA GGTGAGGGTC TCTTTCTTTT 2308CTCTTTTCCT TTTTTTTTTC TTTTTTTGGC AACCTGGCTC TGGCCCAGGC TAGAGTGCAG 2368TGGTGCGATT ATAGCCCGGT GCAGCCTCTA ACTCCTGGGC TCAAGCAATC CAAGTGATCC 2428TCCCACCTCA ACCTTCGGAG TAGCTGGGAT CACAGCTGCA GGCCACGCCC AGCTTCCTCC 2488CCCCGACTCC CCCCCCCCAG AGACACGGTC CCACCATGTT ACCCAGCCTG GTCTCAAACT 2548CCCCAGCTAA AGCAGTCCTC CAGCCTCGGC CTCCCAAAGT ACTGGGATTA CAGGCGTGAG 2608CCCCCACGCT GGCCTGCTTT ACGTATTTTC TTTTGTGCCC CTGCTCACAG TGTTTTAGAG 2668ATGGCTTTCC CAGTGTGTGT TCATTGTAAA CACTTTTGGG AAAGGGCTAA ACATGTGAGG 2728CCTGGAGATA GTTGCTAAGT TGCTAGGAAC ATGTGGTGGG ACTTTCATAT TCTGAAAAAT 2788GTTCTATATT CTCATTTTTC TAAAAGAAAG AAAAAAGGAA ACCCGATTTA TTTCTCCTGA 2848ATCTTTTTAA GTTTGTGTCG TTCCTTAAGC AGAACTAAGC TCAGTATGTG ACCTTACCCG 2908CTAGGTGGTT AATTTATCCA TGCTGGCAGA GGCACTCAGG TACTTGGTAA GCAAATTTCT 2968AAAACTCCAA GTTGCTGCAG CTTGGCATTC TTCTTATTCT AGAGGTCTCT CTGGAAAAGA 3028TGGAGAAAAT GAACAGGACA TGGGGCTCCT GGAAAGAAAG GGCCCGGGAA GTTCAAGGAA 3088GAATAAAGTT GAAATTTTAA AAAAAAA 3115 591 amino acids amino acid linearprotein 2 Val Ala Leu Gln Ile Ala Pro Pro Cys Thr Ser Glu Lys His TyrGlu 1 5 10 15 His Leu Gly Arg Cys Cys Asn Lys Cys Glu Pro Gly Lys TyrMet Ser 20 25 30 Ser Lys Cys Thr Thr Thr Ser Asp Ser Val Cys Leu Pro CysGly Pro 35 40 45 Asp Glu Tyr Leu Asp Ser Trp Asn Glu Glu Asp Lys Cys LeuLeu His 50 55 60 Lys Val Cys Asp Thr Gly Lys Ala Leu Val Ala Val Val AlaGly Asn 65 70 75 80 Ser Thr Thr Pro Arg Arg Cys Ala Cys Thr Ala Gly TyrHis Trp Ser 85 90 95 Gln Asp Cys Glu Cys Cys Arg Arg Asn Thr Glu Cys AlaPro Gly Leu 100 105 110 Gly Ala Gln His Pro Leu Gln Leu Asn Lys Asp ThrVal Cys Lys Pro 115 120 125 Cys Leu Ala Gly Tyr Phe Ser Asp Ala Phe SerSer Thr Asp Lys Cys 130 135 140 Arg Pro Trp Thr Asn Cys Thr Phe Leu GlyLys Arg Val Glu His His 145 150 155 160 Gly Thr Glu Lys Ser Asp Ala ValCys Ser Ser Ser Leu Pro Ala Arg 165 170 175 Lys Pro Pro Asn Glu Pro HisVal Tyr Leu Pro Gly Leu Ile Ile Leu 180 185 190 Leu Leu Phe Ala Ser ValAla Leu Val Ala Ala Ile Ile Phe Gly Val 195 200 205 Cys Tyr Arg Lys LysGly Lys Ala Leu Thr Ala Asn Leu Trp His Trp 210 215 220 Ile Asn Glu AlaCys Gly Arg Leu Ser Gly Asp Lys Glu Ser Ser Gly 225 230 235 240 Asp SerCys Val Ser Thr His Thr Ala Asn Phe Gly Gln Gln Gly Ala 245 250 255 CysGlu Gly Val Leu Leu Leu Thr Leu Glu Glu Lys Thr Phe Pro Glu 260 265 270Asp Met Cys Tyr Pro Asp Gln Gly Gly Val Cys Gln Gly Thr Cys Val 275 280285 Gly Gly Gly Pro Tyr Ala Gln Gly Glu Asp Ala Arg Met Leu Ser Leu 290295 300 Val Ser Lys Thr Glu Ile Glu Glu Asp Ser Phe Arg Gln Met Pro Thr305 310 315 320 Glu Asp Glu Tyr Met Asp Arg Pro Ser Gln Pro Thr Asp GlnLeu Leu 325 330 335 Phe Leu Thr Glu Pro Gly Ser Lys Ser Thr Pro Pro PheSer Glu Pro 340 345 350 Leu Glu Val Gly Glu Asn Asp Ser Leu Ser Gln CysPhe Thr Gly Thr 355 360 365 Gln Ser Thr Val Gly Ser Glu Ser Cys Asn CysThr Glu Pro Leu Cys 370 375 380 Arg Thr Asp Trp Thr Pro Met Ser Ser GluAsn Tyr Leu Gln Lys Glu 385 390 395 400 Val Asp Ser Gly His Cys Pro HisTrp Ala Ala Ser Pro Ser Pro Asn 405 410 415 Trp Ala Asp Val Cys Thr GlyCys Arg Asn Pro Pro Gly Glu Asp Cys 420 425 430 Glu Pro Leu Val Gly SerPro Lys Arg Gly Pro Leu Pro Gln Cys Ala 435 440 445 Tyr Gly Met Gly LeuPro Pro Glu Glu Glu Ala Ser Arg Thr Glu Ala 450 455 460 Arg Asp Gln ProGlu Asp Gly Ala Asp Gly Arg Leu Pro Ser Ser Ala 465 470 475 480 Arg AlaGly Ala Gly Ser Gly Ser Ser Pro Gly Gly Gln Ser Pro Ala 485 490 495 SerGly Asn Val Thr Gly Asn Ser Asn Ser Thr Phe Ile Ser Ser Gly 500 505 510Gln Val Met Asn Phe Lys Gly Asp Ile Ile Val Val Tyr Val Ser Gln 515 520525 Thr Ser Gln Glu Gly Ala Ala Ala Ala Ala Glu Pro Met Gly Arg Pro 530535 540 Val Gln Glu Glu Thr Leu Ala Arg Arg Asp Ser Phe Ala Gly Asn Gly545 550 555 560 Pro Arg Phe Pro Asp Pro Cys Gly Gly Pro Glu Gly Leu ArgGlu Pro 565 570 575 Glu Lys Ala Ser Arg Pro Val Gln Glu Gln Gly Gly AlaLys Ala 580 585 590 1391 base pairs nucleic acid single linear cDNA NONO HOMO SAPIENS BONE-MARROW DERIVED DENDRITIC CELLS 9D-15C CDS 39..13913 CCGCTGAGGC CGCGGCGCCC GCCAGCCTGT CCCGCGCC ATG GCC CCG CGC GCC 53 MetAla Pro Arg Ala 1 5 CGG CGG CGC CGC CCG CTG TTC GCG CTG CTG CTG CTC TGCGCG CTG CTC 101 Arg Arg Arg Arg Pro Leu Phe Ala Leu Leu Leu Leu Cys AlaLeu Leu 10 1520 GCC CGG CTG CAG GTG GCT TTG CAG ATC GCT CCT CCA TGT ACCAGT GAG 149 Ala Arg Leu Gln Val Ala Leu Gln Ile Ala Pro Pro Cys Thr SerGlu 25 30 35 AAG CAT TAT GAG CAT CTG GGA CGG TGC TGT AAC AAA TGT GAA CCAGGA 197 Lys His Tyr Glu His Leu Gly Arg Cys Cys Asn Lys Cys Glu Pro Gly40 45 50 AAG TAC ATG TCT TCT AAA TGC ACT ACT ACC TCT GAC AGT GTA TGT CTG245 Lys Tyr Met Ser Ser Lys Cys Thr Thr Thr Ser Asp Ser Val Cys Leu 556065 CCC TGT GGC CCG GAT GAA TAC TTG GAT AGC TGG AAT GAA GAA GAT AAA 293Pro Cys Gly Pro Asp Glu Tyr Leu Asp Ser Trp Asn Glu Glu Asp Lys 70 75 8085 TGC TTG CTG CAT AAA GTT TGT GAT ACA GGC AAG GCC CTG GTG GCC GTG 341Cys Leu Leu His Lys Val Cys Asp Thr Gly Lys Ala Leu Val Ala Val 90 95100GTC GCC GGC AAC AGC ACG ACC CCC CGG CGC TGC GCG TGC ACG GCT GGG 389 ValAla Gly Asn Ser Thr Thr Pro Arg Arg Cys Ala Cys Thr Ala Gly 1051101150TAC CAC TGG AGC CAG GAC TGC GAG TGC TGC CGC CGC AAC ACC GAG TGC 437 TyrHis Trp Ser Gln Asp Cys Glu Cys Cys Arg Arg Asn Thr Glu Cys 120125130GCG CCG GGC CTG GGC GCC CAG CAC CCG TTG CAG CTC AAC AAG GAC ACA 485 AlaPro Gly Leu Gly Ala Gln His Pro Leu Gln Leu Asn Lys Asp Thr 135 140 145GTG TGC AAA CCT TGC CTT GCA GGC TAC TTC TCT GAT GCC TTT TCC TCC 533 ValCys Lys Pro Cys Leu Ala Gly Tyr Phe Ser Asp Ala Phe Ser Ser 150155160165ACG GAC AAA TGC AGA CCC TGG ACC AAC TGT ACC TTC CTT GGA AAG AGA 581 ThrAsp Lys Cys Arg Pro Trp Thr Asn Cys Thr Phe Leu Gly Lys Arg 170175180GTA GAA CAT CAT GGG ACA GAG AAA TCC GAT GCG GTT TGC AGT TCT TCT 629 ValGlu His His Gly Thr Glu Lys Ser Asp Ala Val Cys Ser Ser Ser 185 190195CTG CCA GCT AGA AAA CCA CCA AAT GAA CCC CAT GTT TAC TTG CCC GGT 677 LeuPro Ala Arg Lys Pro Pro Asn Glu Pro His Val Tyr Leu Pro Gly 200205210TTA ATA ATT CTG CTT CTC TTC GCG TCT GTG GCC CTG GTG GCT GCC ATC 725 LeuIle Ile Leu Leu Leu Phe Ala Ser Val Ala Leu Val Ala Ala Ile 215 220225ATC TTT GGC GTT TGC TAT AGG AAA AAA GGG AAA GCA CTC ACA GCT AAT 773 IlePhe Gly Val Cys Tyr Arg Lys Lys Gly Lys Ala Leu Thr Ala Asn 230235240245TTG TGG CAC TGG ATC AAT GAG GCT TGT GGC CGC CTA AGT GGA GAT AAG 821 LeuTrp His Trp Ile Asn Glu Ala Cys Gly Arg Leu Ser Gly Asp Lys 250255260GAG TCC TCA GGT GAC AGT TGT GTC AGT ACA CAC ACG GCA AAC TTT GGT 869 GluSer Ser Gly Asp Ser Cys Val Ser Thr His Thr Ala Asn Phe Gly 265270275CAG CAG GGA GCA TGT GAA GGT GTC TTA CTG CTG ACT CTG GAG GAG AAG 917 GlnGln Gly Ala Cys Glu Gly Val Leu Leu Leu Thr Leu Glu Glu Lys 280285290ACA TTT CCA GAA GAT ATG TGC TAC CCA GAT CAA GGT GGT GTC TGT CAG 965 ThrPhe Pro Glu Asp Met Cys Tyr Pro Asp Gln Gly Gly Val Cys Gln 295 300305GGC ACG TGT GTA GGA GGT GGT CCC TAC GCA CAA GGC GAA GAT GCC AGG 1013 GlyThr Cys Val Gly Gly Gly Pro Tyr Ala Gln Gly Glu Asp Ala Arg 310315320325ATG CTC TCA TTG GTC AGC AAG ACC GAG ATA GAG GAA GAC AGC TTC AGA 1061 MetLeu Ser Leu Val Ser Lys Thr Glu Ile Glu Glu Asp Ser Phe Arg 330335340CAG ATG CCC ACA GAA GAT GAA TAC ATG GAC AGG CCC TCC CAG CCC ACA 1109 GlnMet Pro Thr Glu Asp Glu Tyr Met Asp Arg Pro Ser Gln Pro Thr 345350355GAC CAG TTA CTG TTC CTC ACT GAG CCT GGA AGC AAA TCC ACA CCT CCT 1157 AspGln Leu Leu Phe Leu Thr Glu Pro Gly Ser Lys Ser Thr Pro Pro 360365370TTC TCT GAA CCC CTG GAG GTG GGG GAG AAT GAC AGT TTA AGC CAG TGC 1205 PheSer Glu Pro Leu Glu Val Gly Glu Asn Asp Ser Leu Ser Gln Cys 375 380385TTC ACG GGG ACA CAG AGC ACA GTG GGT TCA GAA AGC TGC AAC TGC ACT 1253 PheThr Gly Thr Gln Ser Thr Val Gly Ser Glu Ser Cys Asn Cys Thr 390395400405GAG CCC CTG TGC AGG ACT GAT TGG ACT CCC ATG TCC TCT GAA AAC TAC 1301 GluPro Leu Cys Arg Thr Asp Trp Thr Pro Met Ser Ser Glu Asn Tyr 410415420TTG CAA AAA GAG GTG GAC AGT GGC CAT TGC CCG CAC TGG GCA GCC AGC 1349 LeuGln Lys Glu Val Asp Ser Gly His Cys Pro His Trp Ala Ala Ser 425430435CCC AGC CCC AAC TGG GCA GAT GTC TGC ACA GGC TGC CGG AAC 1391 Pro Ser ProAsn Trp Ala Asp Val Cys Thr Gly Cys Arg Asn 440445450 451 amino acidsamino acid linear protein 4 Met Ala Pro Arg Ala Arg Arg Arg Arg Pro LeuPhe Ala Leu Leu Leu 1 5 10 15 Leu Cys Ala Leu Leu Ala Arg Leu Gln ValAla Leu Gln Ile Ala Pro 20 25 30 Pro Cys Thr Ser Glu Lys His Tyr Glu HisLeu Gly Arg Cys Cys Asn 35 40 45 Lys Cys Glu Pro Gly Lys Tyr Met Ser SerLys Cys Thr Thr Thr Ser 50 55 60 Asp Ser Val Cys Leu Pro Cys Gly Pro AspGlu Tyr Leu Asp Ser Trp 65 70 75 80 Asn Glu Glu Asp Lys Cys Leu Leu HisLys Val Cys Asp Thr Gly Lys 85 90 95 Ala Leu Val Ala Val Val Ala Gly AsnSer Thr Thr Pro Arg Arg Cys 100 105 110 Ala Cys Thr Ala Gly Tyr His TrpSer Gln Asp Cys Glu Cys Cys Arg 115 120 125 Arg Asn Thr Glu Cys Ala ProGly Leu Gly Ala Gln His Pro Leu Gln 130 135 140 Leu Asn Lys Asp Thr ValCys Lys Pro Cys Leu Ala Gly Tyr Phe Ser 145 150 155 160 Asp Ala Phe SerSer Thr Asp Lys Cys Arg Pro Trp Thr Asn Cys Thr 165 170 175 Phe Leu GlyLys Arg Val Glu His His Gly Thr Glu Lys Ser Asp Ala 180 185 190 Val CysSer Ser Ser Leu Pro Ala Arg Lys Pro Pro Asn Glu Pro His 195 200 205 ValTyr Leu Pro Gly Leu Ile Ile Leu Leu Leu Phe Ala Ser Val Ala 210 215 220Leu Val Ala Ala Ile Ile Phe Gly Val Cys Tyr Arg Lys Lys Gly Lys 225 230235 240 Ala Leu Thr Ala Asn Leu Trp His Trp Ile Asn Glu Ala Cys Gly Arg245 250 255 Leu Ser Gly Asp Lys Glu Ser Ser Gly Asp Ser Cys Val Ser ThrHis 260 265 270 Thr Ala Asn Phe Gly Gln Gln Gly Ala Cys Glu Gly Val LeuLeu Leu 275 280 285 Thr Leu Glu Glu Lys Thr Phe Pro Glu Asp Met Cys TyrPro Asp Gln 290 295 300 Gly Gly Val Cys Gln Gly Thr Cys Val Gly Gly GlyPro Tyr Ala Gln 305 310 315 320 Gly Glu Asp Ala Arg Met Leu Ser Leu ValSer Lys Thr Glu Ile Glu 325 330 335 Glu Asp Ser Phe Arg Gln Met Pro ThrGlu Asp Glu Tyr Met Asp Arg 340 345 350 Pro Ser Gln Pro Thr Asp Gln LeuLeu Phe Leu Thr Glu Pro Gly Ser 355 360 365 Lys Ser Thr Pro Pro Phe SerGlu Pro Leu Glu Val Gly Glu Asn Asp 370 375 380 Ser Leu Ser Gln Cys PheThr Gly Thr Gln Ser Thr Val Gly Ser Glu 385 390 395 400 Ser Cys Asn CysThr Glu Pro Leu Cys Arg Thr Asp Trp Thr Pro Met 405 410 415 Ser Ser GluAsn Tyr Leu Gln Lys Glu Val Asp Ser Gly His Cys Pro 420 425 430 His TrpAla Ala Ser Pro Ser Pro Asn Trp Ala Asp Val Cys Thr Gly 435 440 445 CysArg Asn 450 3136 base pairs nucleic acid single linear cDNA NO NO HOMOSAPIENS BONE-MARROW DERIVED DENDRITIC CELLS FULL LENGTH RANK CDS39..1886 5 CCGCTGAGGC CGCGGCGCCC GCCAGCCTGT CCCGCGCC ATG GCC CCG CGC GCC53 Met Ala Pro Arg Ala 1 5 CGG CGG CGC CGC CCG CTG TTC GCG CTG CTG CTGCTC TGC GCG CTG CTC 101 Arg Arg Arg Arg Pro Leu Phe Ala Leu Leu Leu LeuCys Ala Leu Leu 10 15 20 GCC CGG CTG CAG GTG GCT TTG CAG ATC GCT CCT CCATGT ACC AGT GAG 149 Ala Arg Leu Gln Val Ala Leu Gln Ile Ala Pro Pro CysThr Ser Glu 25 30 35 AAG CAT TAT GAG CAT CTG GGA CGG TGC TGT AAC AAA TGTGAA CCA GGA 197 Lys His Tyr Glu His Leu Gly Arg Cys Cys Asn Lys Cys GluPro Gly 40 45 50 AAG TAC ATG TCT TCT AAA TGC ACT ACT ACC TCT GAC AGT GTATGT CTG 245 Lys Tyr Met Ser Ser Lys Cys Thr Thr Thr Ser Asp Ser Val CysLeu 55 60 65 CCC TGT GGC CCG GAT GAA TAC TTG GAT AGC TGG AAT GAA GAA GATAAA 293 Pro Cys Gly Pro Asp Glu Tyr Leu Asp Ser Trp Asn Glu Glu Asp Lys70 75 80 85 TGC TTG CTG CAT AAA GTT TGT GAT ACA GGC AAG GCC CTG GTG GCCGTG 341 Cys Leu Leu His Lys Val Cys Asp Thr Gly Lys Ala Leu Val Ala Val90 95 100 GTC GCC GGC AAC AGC ACG ACC CCC CGG CGC TGC GCG TGC ACG GCTGGG 389 Val Ala Gly Asn Ser Thr Thr Pro Arg Arg Cys Ala Cys Thr Ala Gly105 110 115 TAC CAC TGG AGC CAG GAC TGC GAG TGC TGC CGC CGC AAC ACC GAGTGC 437 Tyr His Trp Ser Gln Asp Cys Glu Cys Cys Arg Arg Asn Thr Glu Cys120 125 130 GCG CCG GGC CTG GGC GCC CAG CAC CCG TTG CAG CTC AAC AAG GACACA 485 Ala Pro Gly Leu Gly Ala Gln His Pro Leu Gln Leu Asn Lys Asp Thr135 140 145 GTG TGC AAA CCT TGC CTT GCA GGC TAC TTC TCT GAT GCC TTT TCCTCC 533 Val Cys Lys Pro Cys Leu Ala Gly Tyr Phe Ser Asp Ala Phe Ser Ser150 155 160 165 ACG GAC AAA TGC AGA CCC TGG ACC AAC TGT ACC TTC CTT GGAAAG AGA 581 Thr Asp Lys Cys Arg Pro Trp Thr Asn Cys Thr Phe Leu Gly LysArg 170 175 180 GTA GAA CAT CAT GGG ACA GAG AAA TCC GAT GCG GTT TGC AGTTCT TCT 629 Val Glu His His Gly Thr Glu Lys Ser Asp Ala Val Cys Ser SerSer 185 190 195 CTG CCA GCT AGA AAA CCA CCA AAT GAA CCC CAT GTT TAC TTGCCC GGT 677 Leu Pro Ala Arg Lys Pro Pro Asn Glu Pro His Val Tyr Leu ProGly 200 205 210 TTA ATA ATT CTG CTT CTC TTC GCG TCT GTG GCC CTG GTG GCTGCC ATC 725 Leu Ile Ile Leu Leu Leu Phe Ala Ser Val Ala Leu Val Ala AlaIle 215 220 225 ATC TTT GGC GTT TGC TAT AGG AAA AAA GGG AAA GCA CTC ACAGCT AAT 773 Ile Phe Gly Val Cys Tyr Arg Lys Lys Gly Lys Ala Leu Thr AlaAsn 230 235 240 245 TTG TGG CAC TGG ATC AAT GAG GCT TGT GGC CGC CTA AGTGGA GAT AAG 821 Leu Trp His Trp Ile Asn Glu Ala Cys Gly Arg Leu Ser GlyAsp Lys 250 255 260 GAG TCC TCA GGT GAC AGT TGT GTC AGT ACA CAC ACG GCAAAC TTT GGT 869 Glu Ser Ser Gly Asp Ser Cys Val Ser Thr His Thr Ala AsnPhe Gly 265 270 275 CAG CAG GGA GCA TGT GAA GGT GTC TTA CTG CTG ACT CTGGAG GAG AAG 917 Gln Gln Gly Ala Cys Glu Gly Val Leu Leu Leu Thr Leu GluGlu Lys 280 285 290 ACA TTT CCA GAA GAT ATG TGC TAC CCA GAT CAA GGT GGTGTC TGT CAG 965 Thr Phe Pro Glu Asp Met Cys Tyr Pro Asp Gln Gly Gly ValCys Gln 295 300 305 GGC ACG TGT GTA GGA GGT GGT CCC TAC GCA CAA GGC GAAGAT GCC AGG 1013 Gly Thr Cys Val Gly Gly Gly Pro Tyr Ala Gln Gly Glu AspAla Arg 310 315 320 325 ATG CTC TCA TTG GTC AGC AAG ACC GAG ATA GAG GAAGAC AGC TTC AGA 1061 Met Leu Ser Leu Val Ser Lys Thr Glu Ile Glu Glu AspSer Phe Arg 330 335 340 CAG ATG CCC ACA GAA GAT GAA TAC ATG GAC AGG CCCTCC CAG CCC ACA 1109 Gln Met Pro Thr Glu Asp Glu Tyr Met Asp Arg Pro SerGln Pro Thr 345 350 355 GAC CAG TTA CTG TTC CTC ACT GAG CCT GGA AGC AAATCC ACA CCT CCT 1157 Asp Gln Leu Leu Phe Leu Thr Glu Pro Gly Ser Lys SerThr Pro Pro 360 365 370 TTC TCT GAA CCC CTG GAG GTG GGG GAG AAT GAC AGTTTA AGC CAG TGC 1205 Phe Ser Glu Pro Leu Glu Val Gly Glu Asn Asp Ser LeuSer Gln Cys 375 380 385 TTC ACG GGG ACA CAG AGC ACA GTG GGT TCA GAA AGCTGC AAC TGC ACT 1253 Phe Thr Gly Thr Gln Ser Thr Val Gly Ser Glu Ser CysAsn Cys Thr 390 395 400 405 GAG CCC CTG TGC AGG ACT GAT TGG ACT CCC ATGTCC TCT GAA AAC TAC 1301 Glu Pro Leu Cys Arg Thr Asp Trp Thr Pro Met SerSer Glu Asn Tyr 410 415 420 TTG CAA AAA GAG GTG GAC AGT GGC CAT TGC CCGCAC TGG GCA GCC AGC 1349 Leu Gln Lys Glu Val Asp Ser Gly His Cys Pro HisTrp Ala Ala Ser 425 430 435 CCC AGC CCC AAC TGG GCA GAT GTC TGC ACA GGCTGC CGG AAC CCT CCT 1397 Pro Ser Pro Asn Trp Ala Asp Val Cys Thr Gly CysArg Asn Pro Pro 440 445 450 GGG GAG GAC TGT GAA CCC CTC GTG GGT TCC CCAAAA CGT GGA CCC TTG 1445 Gly Glu Asp Cys Glu Pro Leu Val Gly Ser Pro LysArg Gly Pro Leu 455 460 465 CCC CAG TGC GCC TAT GGC ATG GGC CTT CCC CCTGAA GAA GAA GCC AGC 1493 Pro Gln Cys Ala Tyr Gly Met Gly Leu Pro Pro GluGlu Glu Ala Ser 470 475 480 485 AGG ACG GAG GCC AGA GAC CAG CCC GAG GATGGG GCT GAT GGG AGG CTC 1541 Arg Thr Glu Ala Arg Asp Gln Pro Glu Asp GlyAla Asp Gly Arg Leu 490 495 500 CCA AGC TCA GCG AGG GCA GGT GCC GGG TCTGGA AGC TCC CCT GGT GGC 1589 Pro Ser Ser Ala Arg Ala Gly Ala Gly Ser GlySer Ser Pro Gly Gly 505 510 515 CAG TCC CCT GCA TCT GGA AAT GTG ACT GGAAAC AGT AAC TCC ACG TTC 1637 Gln Ser Pro Ala Ser Gly Asn Val Thr Gly AsnSer Asn Ser Thr Phe 520 525 530 ATC TCC AGC GGG CAG GTG ATG AAC TTC AAGGGC GAC ATC ATC GTG GTC 1685 Ile Ser Ser Gly Gln Val Met Asn Phe Lys GlyAsp Ile Ile Val Val 535 540 545 TAC GTC AGC CAG ACC TCG CAG GAG GGC GCGGCG GCG GCT GCG GAG CCC 1733 Tyr Val Ser Gln Thr Ser Gln Glu Gly Ala AlaAla Ala Ala Glu Pro 550 555 560 565 ATG GGC CGC CCG GTG CAG GAG GAG ACCCTG GCG CGC CGA GAC TCC TTC 1781 Met Gly Arg Pro Val Gln Glu Glu Thr LeuAla Arg Arg Asp Ser Phe 570 575 580 GCG GGG AAC GGC CCG CGC TTC CCG GACCCG TGC GGC GGC CCC GAG GGG 1829 Ala Gly Asn Gly Pro Arg Phe Pro Asp ProCys Gly Gly Pro Glu Gly 585 590 595 CTG CGG GAG CCG GAG AAG GCC TCG AGGCCG GTG CAG GAG CAA GGC GGG 1877 Leu Arg Glu Pro Glu Lys Ala Ser Arg ProVal Gln Glu Gln Gly Gly 600 605 610 GCC AAG GCT TGAGCGCCCC CCATGGCTGGGAGCCCGAAG CTCGGAGCCA 1926 Ala Lys Ala 615 GGGCTCGCGA GGGCAGCACCGCAGCCTCTG CCCCAGCCCC GGCCACCCAG GGATCGATCG 1986 GTACAGTCGA GGAAGACCACCCGGCATTCT CTGCCCACTT TGCCTTCCAG GAAATGGGCT 2046 TTTCAGGAAG TGAATTGATGAGGACTGTCC CCATGCCCAC GGATGCTCAG CAGCCCGCCG 2106 CACTGGGGCA GATGTCTCCCCTGCCACTCC TCAAACTCGC AGCAGTAATT TGTGGCACTA 2166 TGACAGCTAT TTTTATGACTATCCTGTTCT GTGGGGGGGG GGTCTATGTT TTCCCCCCAT 2226 ATTTGTATTC CTTTTCATAACTTTTCTTGA TATCTTTCCT CCCTCTTTTT TAATGTAAAG 2286 GTTTTCTCAA AAATTCTCCTAAAGGTGAGG GTCTCTTTCT TTTCTCTTTT CCTTTTTTTT 2346 TTCTTTTTTT GGCAACCTGGCTCTGGCCCA GGCTAGAGTG CAGTGGTGCG ATTATAGCCC 2406 GGTGCAGCCT CTAACTCCTGGGCTCAAGCA ATCCAAGTGA TCCTCCCACC TCAACCTTCG 2466 GAGTAGCTGG GATCACAGCTGCAGGCCACG CCCAGCTTCC TCCCCCCGAC TCCCCCCCCC 2526 CAGAGACACG GTCCCACCATGTTACCCAGC CTGGTCTCAA ACTCCCCAGC TAAAGCAGTC 2586 CTCCAGCCTC GGCCTCCCAAAGTACTGGGA TTACAGGCGT GAGCCCCCAC GCTGGCCTGC 2646 TTTACGTATT TTCTTTTGTGCCCCTGCTCA CAGTGTTTTA GAGATGGCTT TCCCAGTGTG 2706 TGTTCATTGT AAACACTTTTGGGAAAGGGC TAAACATGTG AGGCCTGGAG ATAGTTGCTA 2766 AGTTGCTAGG AACATGTGGTGGGACTTTCA TATTCTGAAA AATGTTCTAT ATTCTCATTT 2826 TTCTAAAAGA AAGAAAAAAGGAAACCCGAT TTATTTCTCC TGAATCTTTT TAAGTTTGTG 2886 TCGTTCCTTA AGCAGAACTAAGCTCAGTAT GTGACCTTAC CCGCTAGGTG GTTAATTTAT 2946 CCATGCTGGC AGAGGCACTCAGGTACTTGG TAAGCAAATT TCTAAAACTC CAAGTTGCTG 3006 CAGCTTGGCA TTCTTCTTATTCTAGAGGTC TCTCTGGAAA AGATGGAGAA AATGAACAGG 3066 ACATGGGGCT CCTGGAAAGAAAGGGCCCGG GAAGTTCAAG GAAGAATAAA GTTGAAATTT 3126 TAAAAAAAAA 3136 616amino acids amino acid linear protein 6 Met Ala Pro Arg Ala Arg Arg ArgArg Pro Leu Phe Ala Leu Leu Leu 1 5 10 15 Leu Cys Ala Leu Leu Ala ArgLeu Gln Val Ala Leu Gln Ile Ala Pro 20 25 30 Pro Cys Thr Ser Glu Lys HisTyr Glu His Leu Gly Arg Cys Cys Asn 35 40 45 Lys Cys Glu Pro Gly Lys TyrMet Ser Ser Lys Cys Thr Thr Thr Ser 50 55 60 Asp Ser Val Cys Leu Pro CysGly Pro Asp Glu Tyr Leu Asp Ser Trp 65 70 75 80 Asn Glu Glu Asp Lys CysLeu Leu His Lys Val Cys Asp Thr Gly Lys 85 90 95 Ala Leu Val Ala Val ValAla Gly Asn Ser Thr Thr Pro Arg Arg Cys 100 105 110 Ala Cys Thr Ala GlyTyr His Trp Ser Gln Asp Cys Glu Cys Cys Arg 115 120 125 Arg Asn Thr GluCys Ala Pro Gly Leu Gly Ala Gln His Pro Leu Gln 130 135 140 Leu Asn LysAsp Thr Val Cys Lys Pro Cys Leu Ala Gly Tyr Phe Ser 145 150 155 160 AspAla Phe Ser Ser Thr Asp Lys Cys Arg Pro Trp Thr Asn Cys Thr 165 170 175Phe Leu Gly Lys Arg Val Glu His His Gly Thr Glu Lys Ser Asp Ala 180 185190 Val Cys Ser Ser Ser Leu Pro Ala Arg Lys Pro Pro Asn Glu Pro His 195200 205 Val Tyr Leu Pro Gly Leu Ile Ile Leu Leu Leu Phe Ala Ser Val Ala210 215 220 Leu Val Ala Ala Ile Ile Phe Gly Val Cys Tyr Arg Lys Lys GlyLys 225 230 235 240 Ala Leu Thr Ala Asn Leu Trp His Trp Ile Asn Glu AlaCys Gly Arg 245 250 255 Leu Ser Gly Asp Lys Glu Ser Ser Gly Asp Ser CysVal Ser Thr His 260 265 270 Thr Ala Asn Phe Gly Gln Gln Gly Ala Cys GluGly Val Leu Leu Leu 275 280 285 Thr Leu Glu Glu Lys Thr Phe Pro Glu AspMet Cys Tyr Pro Asp Gln 290 295 300 Gly Gly Val Cys Gln Gly Thr Cys ValGly Gly Gly Pro Tyr Ala Gln 305 310 315 320 Gly Glu Asp Ala Arg Met LeuSer Leu Val Ser Lys Thr Glu Ile Glu 325 330 335 Glu Asp Ser Phe Arg GlnMet Pro Thr Glu Asp Glu Tyr Met Asp Arg 340 345 350 Pro Ser Gln Pro ThrAsp Gln Leu Leu Phe Leu Thr Glu Pro Gly Ser 355 360 365 Lys Ser Thr ProPro Phe Ser Glu Pro Leu Glu Val Gly Glu Asn Asp 370 375 380 Ser Leu SerGln Cys Phe Thr Gly Thr Gln Ser Thr Val Gly Ser Glu 385 390 395 400 SerCys Asn Cys Thr Glu Pro Leu Cys Arg Thr Asp Trp Thr Pro Met 405 410 415Ser Ser Glu Asn Tyr Leu Gln Lys Glu Val Asp Ser Gly His Cys Pro 420 425430 His Trp Ala Ala Ser Pro Ser Pro Asn Trp Ala Asp Val Cys Thr Gly 435440 445 Cys Arg Asn Pro Pro Gly Glu Asp Cys Glu Pro Leu Val Gly Ser Pro450 455 460 Lys Arg Gly Pro Leu Pro Gln Cys Ala Tyr Gly Met Gly Leu ProPro 465 470 475 480 Glu Glu Glu Ala Ser Arg Thr Glu Ala Arg Asp Gln ProGlu Asp Gly 485 490 495 Ala Asp Gly Arg Leu Pro Ser Ser Ala Arg Ala GlyAla Gly Ser Gly 500 505 510 Ser Ser Pro Gly Gly Gln Ser Pro Ala Ser GlyAsn Val Thr Gly Asn 515 520 525 Ser Asn Ser Thr Phe Ile Ser Ser Gly GlnVal Met Asn Phe Lys Gly 530 535 540 Asp Ile Ile Val Val Tyr Val Ser GlnThr Ser Gln Glu Gly Ala Ala 545 550 555 560 Ala Ala Ala Glu Pro Met GlyArg Pro Val Gln Glu Glu Thr Leu Ala 565 570 575 Arg Arg Asp Ser Phe AlaGly Asn Gly Pro Arg Phe Pro Asp Pro Cys 580 585 590 Gly Gly Pro Glu GlyLeu Arg Glu Pro Glu Lys Ala Ser Arg Pro Val 595 600 605 Gln Glu Gln GlyGly Ala Lys Ala 610 615 8 amino acids amino acid Not Relevant linearpeptide FLAG_ peptide 7 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 232 aminoacids amino acid Not Relevant linear protein Human IgG1 Fc mutein 8 GluPro Arg Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 20 25 30Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 7580 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 9095 Asp Trp Leu Asn Gly Lys Asp Tyr Lys Cys Lys Val Ser Asn Lys Ala 100105 110 Leu Pro Ala Pro Met Gln Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu LeuThr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe TyrPro Arg 145 150 155 160 His Ile Ala Val Glu Trp Glu Ser Asn Gly Gln ProGlu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp GlySer Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg TrpGln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala LeuHis Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys225 230 31 amino acids amino acid Not Relevant linear peptide NO NO CMV(R2780 Leader) Met1-Arg28 is the actual leader peptide; Arg29strengthens the furin cleavage site; nucleotides encoding eThr30 andSer31 add a Spe1 site. 9 Met Ala Arg Arg Leu Trp Ile Leu Ser Leu Leu AlaVal Thr Leu Thr 1 5 10 15 Val Ala Leu Ala Ala Pro Ser Gln Lys Ser LysArg Arg Thr Ser 20 25 30 1630 base pairs nucleic acid single linear cDNANO NO Mus musculus <Unknown> RANKL CDS 3..884 10 CC GGC GTC CCA CAC GAGGGT CCG CTG CAC CCC GCG CCT TCT GCA CCG 47 Gly Val Pro His Glu Gly ProLeu His Pro Ala Pro Ser Ala Pro 1 5 10 15 GCT CCG GCG CCG CCA CCC GCCGCC TCC CGC TCC ATG TTC CTG GCC CTC 95 Ala Pro Ala Pro Pro Pro Ala AlaSer Arg Ser Met Phe Leu Ala Leu 20 25 30 CTG GGG CTG GGA CTG GGC CAG GTGGTC TGC AGC ATC GCT CTG TTC CTG 143 Leu Gly Leu Gly Leu Gly Gln Val ValCys Ser Ile Ala Leu Phe Leu 35 40 45 TAC TTT CGA GCG CAG ATG GAT CCT AACAGA ATA TCA GAA GAC AGC ACT 191 Tyr Phe Arg Ala Gln Met Asp Pro Asn ArgIle Ser Glu Asp Ser Thr 50 55 60 CAC TGC TTT TAT AGA ATC CTG AGA CTC CATGAA AAC GCA GAT TTG CAG 239 His Cys Phe Tyr Arg Ile Leu Arg Leu His GluAsn Ala Asp Leu Gln 65 70 75 GAC TCG ACT CTG GAG AGT GAA GAC ACA CTA CCTGAC TCC TGC AGG AGG 287 Asp Ser Thr Leu Glu Ser Glu Asp Thr Leu Pro AspSer Cys Arg Arg 80 85 90 95 ATG AAA CAA GCC TTT CAG GGG GCC GTG CAG AAGGAA CTG CAA CAC ATT 335 Met Lys Gln Ala Phe Gln Gly Ala Val Gln Lys GluLeu Gln His Ile 100 105 110 GTG GGG CCA CAG CGC TTC TCA GGA GCT CCA GCTATG ATG GAA GGC TCA 383 Val Gly Pro Gln Arg Phe Ser Gly Ala Pro Ala MetMet Glu Gly Ser 115 120 125 TGG TTG GAT GTG GCC CAG CGA GGC AAG CCT GAGGCC CAG CCA TTT GCA 431 Trp Leu Asp Val Ala Gln Arg Gly Lys Pro Glu AlaGln Pro Phe Ala 130 135 140 CAC CTC ACC ATC AAT GCT GCC AGC ATC CCA TCGGGT TCC CAT AAA GTC 479 His Leu Thr Ile Asn Ala Ala Ser Ile Pro Ser GlySer His Lys Val 145 150 155 ACT CTG TCC TCT TGG TAC CAC GAT CGA GGC TGGGCC AAG ATC TCT AAC 527 Thr Leu Ser Ser Trp Tyr His Asp Arg Gly Trp AlaLys Ile Ser Asn 160 165 170 175 ATG ACG TTA AGC AAC GGA AAA CTA AGG GTTAAC CAA GAT GGC TTC TAT 575 Met Thr Leu Ser Asn Gly Lys Leu Arg Val AsnGln Asp Gly Phe Tyr 180 185 190 TAC CTG TAC GCC AAC ATT TGC TTT CGG CATCAT GAA ACA TCG GGA AGC 623 Tyr Leu Tyr Ala Asn Ile Cys Phe Arg His HisGlu Thr Ser Gly Ser 195 200 205 GTA CCT ACA GAC TAT CTT CAG CTG ATG GTGTAT GTC GTT AAA ACC AGC 671 Val Pro Thr Asp Tyr Leu Gln Leu Met Val TyrVal Val Lys Thr Ser 210 215 220 ATC AAA ATC CCA AGT TCT CAT AAC CTG ATGAAA GGA GGG AGC ACG AAA 719 Ile Lys Ile Pro Ser Ser His Asn Leu Met LysGly Gly Ser Thr Lys 225 230 235 AAC TGG TCG GGC AAT TCT GAA TTC CAC TTTTAT TCC ATA AAT GTT GGG 767 Asn Trp Ser Gly Asn Ser Glu Phe His Phe TyrSer Ile Asn Val Gly 240 245 250 255 GGA TTT TTC AAG CTC CGA GCT GGT GAAGAA ATT AGC ATT CAG GTG TCC 815 Gly Phe Phe Lys Leu Arg Ala Gly Glu GluIle Ser Ile Gln Val Ser 260 265 270 AAC CCT TCC CTG CTG GAT CCG GAT CAAGAT GCG ACG TAC TTT GGG GCT 863 Asn Pro Ser Leu Leu Asp Pro Asp Gln AspAla Thr Tyr Phe Gly Ala 275 280 285 TTC AAA GTT CAG GAC ATA GACTGAGACTCAT TTCGTGGAAC ATTAGCATGG 914 Phe Lys Val Gln Asp Ile Asp 290ATGTCCTAGA TGTTTGGAAA CTTCTTAAAA AATGGATGAT GTCTATACAT GTGTAAGACT 974ACTAAGAGAC ATGGCCCACG GTGTATGAAA CTCACAGCCC TCTCTCTTGA GCCTGTACAG 1034GTTGTGTATA TGTAAAGTCC ATAGGTGATG TTAGATTCAT GGTGATTACA CAACGGTTTT 1094ACAATTTTGT AATGATTTCC TAGAATTGAA CCAGATTGGG AGAGGTATTC CGATGCTTAT 1154GAAAAACTTA CACGTGAGCT ATGGAAGGGG GTCACAGTCT CTGGGTCTAA CCCCTGGACA 1214TGTGCCACTG AGAACCTTGA AATTAAGAGG ATGCCATGTC ATTGCAAAGA AATGATAGTG 1274TGAAGGGTTA AGTTCTTTTG AATTGTTACA TTGCGCTGGG ACCTGCAAAT AAGTTCTTTT 1334TTTCTAATGA GGAGAGAAAA ATATATGTAT TTTTATATAA TGTCTAAAGT TATATTTCAG 1394GTGTAATGTT TTCTGTGCAA AGTTTTGTAA ATTATATTTG TGCTATAGTA TTTGATTCAA 1454AATATTTAAA AATGTCTCAC TGTTGACATA TTTAATGTTT TAAATGTACA GATGTATTTA 1514ACTGGTGCAC TTTGTAATTC CCCTGAAGGT ACTCGTAGCT AAGGGGGCAG AATACTGTTT 1574CTGGTGACCA CATGTAGTTT ATTTCTTTAT TCTTTTTAAC TTAATAGAGT CTTCAG 1630 294amino acids amino acid linear protein 11 Gly Val Pro His Glu Gly Pro LeuHis Pro Ala Pro Ser Ala Pro Ala 1 5 10 15 Pro Ala Pro Pro Pro Ala AlaSer Arg Ser Met Phe Leu Ala Leu Leu 20 25 30 Gly Leu Gly Leu Gly Gln ValVal Cys Ser Ile Ala Leu Phe Leu Tyr 35 40 45 Phe Arg Ala Gln Met Asp ProAsn Arg Ile Ser Glu Asp Ser Thr His 50 55 60 Cys Phe Tyr Arg Ile Leu ArgLeu His Glu Asn Ala Asp Leu Gln Asp 65 70 75 80 Ser Thr Leu Glu Ser GluAsp Thr Leu Pro Asp Ser Cys Arg Arg Met 85 90 95 Lys Gln Ala Phe Gln GlyAla Val Gln Lys Glu Leu Gln His Ile Val 100 105 110 Gly Pro Gln Arg PheSer Gly Ala Pro Ala Met Met Glu Gly Ser Trp 115 120 125 Leu Asp Val AlaGln Arg Gly Lys Pro Glu Ala Gln Pro Phe Ala His 130 135 140 Leu Thr IleAsn Ala Ala Ser Ile Pro Ser Gly Ser His Lys Val Thr 145 150 155 160 LeuSer Ser Trp Tyr His Asp Arg Gly Trp Ala Lys Ile Ser Asn Met 165 170 175Thr Leu Ser Asn Gly Lys Leu Arg Val Asn Gln Asp Gly Phe Tyr Tyr 180 185190 Leu Tyr Ala Asn Ile Cys Phe Arg His His Glu Thr Ser Gly Ser Val 195200 205 Pro Thr Asp Tyr Leu Gln Leu Met Val Tyr Val Val Lys Thr Ser Ile210 215 220 Lys Ile Pro Ser Ser His Asn Leu Met Lys Gly Gly Ser Thr LysAsn 225 230 235 240 Trp Ser Gly Asn Ser Glu Phe His Phe Tyr Ser Ile AsnVal Gly Gly 245 250 255 Phe Phe Lys Leu Arg Ala Gly Glu Glu Ile Ser IleGln Val Ser Asn 260 265 270 Pro Ser Leu Leu Asp Pro Asp Gln Asp Ala ThrTyr Phe Gly Ala Phe 275 280 285 Lys Val Gln Asp Ile Asp 290 954 basepairs nucleic acid single linear cDNA NO NO Homo sapiens <Unknown>huRANKL (full length) CDS 1..951 12 ATG CGC CGC GCC AGC AGA GAC TAC ACCAAG TAC CTG CGT GGC TCG GAG 48 Met Arg Arg Ala Ser Arg Asp Tyr Thr LysTyr Leu Arg Gly Ser Glu 1 5 10 15 GAG ATG GGC GGC GGC CCC GGA GCC CCGCAC GAG GGC CCC CTG CAC GCC 96 Glu Met Gly Gly Gly Pro Gly Ala Pro HisGlu Gly Pro Leu His Ala 20 25 30 CCG CCG CCG CCT GCG CCG CAC CAG CCC CCCGCC GCC TCC CGC TCC ATG 144 Pro Pro Pro Pro Ala Pro His Gln Pro Pro AlaAla Ser Arg Ser Met 35 40 45 TTC GTG GCC CTC CTG GGG CTG GGG CTG GGC CAGGTT GTC TGC AGC GTC 192 Phe Val Ala Leu Leu Gly Leu Gly Leu Gly Gln ValVal Cys Ser Val 50 55 60 GCC CTG TTC TTC TAT TTC AGA GCG CAG ATG GAT CCTAAT AGA ATA TCA 240 Ala Leu Phe Phe Tyr Phe Arg Ala Gln Met Asp Pro AsnArg Ile Ser 65 70 75 80 GAA GAT GGC ACT CAC TGC ATT TAT AGA ATT TTG AGACTC CAT GAA AAT 288 Glu Asp Gly Thr His Cys Ile Tyr Arg Ile Leu Arg LeuHis Glu Asn 85 90 95 GCA GAT TTT CAA GAC ACA ACT CTG GAG AGT CAA GAT ACAAAA TTA ATA 336 Ala Asp Phe Gln Asp Thr Thr Leu Glu Ser Gln Asp Thr LysLeu Ile 100 105 110 CCT GAT TCA TGT AGG AGA ATT AAA CAG GCC TTT CAA GGAGCT GTG CAA 384 Pro Asp Ser Cys Arg Arg Ile Lys Gln Ala Phe Gln Gly AlaVal Gln 115 120 125 AAG GAA TTA CAA CAT ATC GTT GGA TCA CAG CAC ATC AGAGCA GAG AAA 432 Lys Glu Leu Gln His Ile Val Gly Ser Gln His Ile Arg AlaGlu Lys 130 135 140 GCG ATG GTG GAT GGC TCA TGG TTA GAT CTG GCC AAG AGGAGC AAG CTT 480 Ala Met Val Asp Gly Ser Trp Leu Asp Leu Ala Lys Arg SerLys Leu 145 150 155 160 GAA GCT CAG CCT TTT GCT CAT CTC ACT ATT AAT GCCACC GAC ATC CCA 528 Glu Ala Gln Pro Phe Ala His Leu Thr Ile Asn Ala ThrAsp Ile Pro 165 170 175 TCT GGT TCC CAT AAA GTG AGT CTG TCC TCT TGG TACCAT GAT CGG GGT 576 Ser Gly Ser His Lys Val Ser Leu Ser Ser Trp Tyr HisAsp Arg Gly 180 185 190 TGG GCC AAG ATC TCC AAC ATG ACT TTT AGC AAT GGAAAA CTA ATA GTT 624 Trp Ala Lys Ile Ser Asn Met Thr Phe Ser Asn Gly LysLeu Ile Val 195 200 205 AAT CAG GAT GGC TTT TAT TAC CTG TAT GCC AAC ATTTGC TTT CGA CAT 672 Asn Gln Asp Gly Phe Tyr Tyr Leu Tyr Ala Asn Ile CysPhe Arg His 210 215 220 CAT GAA ACT TCA GGA GAC CTA GCT ACA GAG TAT CTTCAA CTA ATG GTG 720 His Glu Thr Ser Gly Asp Leu Ala Thr Glu Tyr Leu GlnLeu Met Val 225 230 235 240 TAC GTC ACT AAA ACC AGC ATC AAA ATC CCA AGTTCT CAT ACC CTG ATG 768 Tyr Val Thr Lys Thr Ser Ile Lys Ile Pro Ser SerHis Thr Leu Met 245 250 255 AAA GGA GGA AGC ACC AAG TAT TGG TCA GGG AATTCT GAA TTC CAT TTT 816 Lys Gly Gly Ser Thr Lys Tyr Trp Ser Gly Asn SerGlu Phe His Phe 260 265 270 TAT TCC ATA AAC GTT GGT GGA TTT TTT AAG TTACGG TCT GGA GAG GAA 864 Tyr Ser Ile Asn Val Gly Gly Phe Phe Lys Leu ArgSer Gly Glu Glu 275 280 285 ATC AGC ATC GAG GTC TCC AAC CCC TCC TTA CTGGAT CCG GAT CAG GAT 912 Ile Ser Ile Glu Val Ser Asn Pro Ser Leu Leu AspPro Asp Gln Asp 290 295 300 GCA ACA TAC TTT GGG GCT TTT AAA GTT CGA GATATA GAT TGA 954 Ala Thr Tyr Phe Gly Ala Phe Lys Val Arg Asp Ile Asp 305310 315 317 amino acids amino acid linear protein 13 Met Arg Arg Ala SerArg Asp Tyr Thr Lys Tyr Leu Arg Gly Ser Glu 1 5 10 15 Glu Met Gly GlyGly Pro Gly Ala Pro His Glu Gly Pro Leu His Ala 20 25 30 Pro Pro Pro ProAla Pro His Gln Pro Pro Ala Ala Ser Arg Ser Met 35 40 45 Phe Val Ala LeuLeu Gly Leu Gly Leu Gly Gln Val Val Cys Ser Val 50 55 60 Ala Leu Phe PheTyr Phe Arg Ala Gln Met Asp Pro Asn Arg Ile Ser 65 70 75 80 Glu Asp GlyThr His Cys Ile Tyr Arg Ile Leu Arg Leu His Glu Asn 85 90 95 Ala Asp PheGln Asp Thr Thr Leu Glu Ser Gln Asp Thr Lys Leu Ile 100 105 110 Pro AspSer Cys Arg Arg Ile Lys Gln Ala Phe Gln Gly Ala Val Gln 115 120 125 LysGlu Leu Gln His Ile Val Gly Ser Gln His Ile Arg Ala Glu Lys 130 135 140Ala Met Val Asp Gly Ser Trp Leu Asp Leu Ala Lys Arg Ser Lys Leu 145 150155 160 Glu Ala Gln Pro Phe Ala His Leu Thr Ile Asn Ala Thr Asp Ile Pro165 170 175 Ser Gly Ser His Lys Val Ser Leu Ser Ser Trp Tyr His Asp ArgGly 180 185 190 Trp Ala Lys Ile Ser Asn Met Thr Phe Ser Asn Gly Lys LeuIle Val 195 200 205 Asn Gln Asp Gly Phe Tyr Tyr Leu Tyr Ala Asn Ile CysPhe Arg His 210 215 220 His Glu Thr Ser Gly Asp Leu Ala Thr Glu Tyr LeuGln Leu Met Val 225 230 235 240 Tyr Val Thr Lys Thr Ser Ile Lys Ile ProSer Ser His Thr Leu Met 245 250 255 Lys Gly Gly Ser Thr Lys Tyr Trp SerGly Asn Ser Glu Phe His Phe 260 265 270 Tyr Ser Ile Asn Val Gly Gly PhePhe Lys Leu Arg Ser Gly Glu Glu 275 280 285 Ile Ser Ile Glu Val Ser AsnPro Ser Leu Leu Asp Pro Asp Gln Asp 290 295 300 Ala Thr Tyr Phe Gly AlaPhe Lys Val Arg Asp Ile Asp 305 310 315 1878 base pairs nucleic acidsingle linear cDNA NO NO Murine Murine Fetal Liver Epithelium muRANK CDS1..1875 14 ATG GCC CCG CGC GCC CGG CGG CGC CGC CAG CTG CCC GCG CCG CTGCTG 48 Met Ala Pro Arg Ala Arg Arg Arg Arg Gln Leu Pro Ala Pro Leu Leu 15 10 15 GCG CTC TGC GTG CTG CTC GTT CCA CTG CAG GTG ACT CTC CAG GTC ACT96 Ala Leu Cys Val Leu Leu Val Pro Leu Gln Val Thr Leu Gln Val Thr 20 2530 CCT CCA TGC ACC CAG GAG AGG CAT TAT GAG CAT CTC GGA CGG TGT TGC 144Pro Pro Cys Thr Gln Glu Arg His Tyr Glu His Leu Gly Arg Cys Cys 35 40 45AGC AGA TGC GAA CCA GGA AAG TAC CTG TCC TCT AAG TGC ACT CCT ACC 192 SerArg Cys Glu Pro Gly Lys Tyr Leu Ser Ser Lys Cys Thr Pro Thr 50 55 60 TCCGAC AGT GTG TGT CTG CCC TGT GGC CCC GAT GAG TAC TTG GAC ACC 240 Ser AspSer Val Cys Leu Pro Cys Gly Pro Asp Glu Tyr Leu Asp Thr 65 70 75 80 TGGAAT GAA GAA GAT AAA TGC TTG CTG CAT AAA GTC TGT GAT GCA GGC 288 Trp AsnGlu Glu Asp Lys Cys Leu Leu His Lys Val Cys Asp Ala Gly 85 90 95 AAG GCCCTG GTG GCG GTG GAT CCT GGC AAC CAC ACG GCC CCG CGT CGC 336 Lys Ala LeuVal Ala Val Asp Pro Gly Asn His Thr Ala Pro Arg Arg 100 105 110 TGT GCTTGC ACG GCT GGC TAC CAC TGG AAC TCA GAC TGC GAG TGC TGC 384 Cys Ala CysThr Ala Gly Tyr His Trp Asn Ser Asp Cys Glu Cys Cys 115 120 125 CGC AGGAAC ACG GAG TGT GCA CCT GGC TTC GGA GCT CAG CAT CCC TTG 432 Arg Arg AsnThr Glu Cys Ala Pro Gly Phe Gly Ala Gln His Pro Leu 130 135 140 CAG CTCAAC AAG GAT ACG GTG TGC ACA CCC TGC CTC CTG GGC TTC TTC 480 Gln Leu AsnLys Asp Thr Val Cys Thr Pro Cys Leu Leu Gly Phe Phe 145 150 155 160 TCAGAT GTC TTT TCG TCC ACA GAC AAA TGC AAA CCT TGG ACC AAC TGC 528 Ser AspVal Phe Ser Ser Thr Asp Lys Cys Lys Pro Trp Thr Asn Cys 165 170 175 ACCCTC CTT GGA AAG CTA GAA GCA CAC CAG GGG ACA ACG GAA TCA GAT 576 Thr LeuLeu Gly Lys Leu Glu Ala His Gln Gly Thr Thr Glu Ser Asp 180 185 190 GTGGTC TGC AGC TCT TCC ATG ACA CTG AGG AGA CCA CCC AAG GAG GCC 624 Val ValCys Ser Ser Ser Met Thr Leu Arg Arg Pro Pro Lys Glu Ala 195 200 205 CAGGCT TAC CTG CCC AGT CTC ATC GTT CTG CTC CTC TTC ATC TCT GTG 672 Gln AlaTyr Leu Pro Ser Leu Ile Val Leu Leu Leu Phe Ile Ser Val 210 215 220 GTAGTA GTG GCT GCC ATC ATC TTC GGC GTT TAC TAC AGG AAG GGA GGG 720 Val ValVal Ala Ala Ile Ile Phe Gly Val Tyr Tyr Arg Lys Gly Gly 225 230 235 240AAA GCG CTG ACA GCT AAT TTG TGG AAT TGG GTC AAT GAT GCT TGC AGT 768 LysAla Leu Thr Ala Asn Leu Trp Asn Trp Val Asn Asp Ala Cys Ser 245 250 255AGT CTA AGT GGA AAT AAG GAG TCC TCA GGG GAC CGT TGT GCT GGT TCC 816 SerLeu Ser Gly Asn Lys Glu Ser Ser Gly Asp Arg Cys Ala Gly Ser 260 265 270CAC TCG GCA ACC TCC AGT CAG CAA GAA GTG TGT GAA GGT ATC TTA CTA 864 HisSer Ala Thr Ser Ser Gln Gln Glu Val Cys Glu Gly Ile Leu Leu 275 280 285ATG ACT CGG GAG GAG AAG ATG GTT CCA GAA GAC GGT GCT GGA GTC TGT 912 MetThr Arg Glu Glu Lys Met Val Pro Glu Asp Gly Ala Gly Val Cys 290 295 300GGG CCT GTG TGT GCG GCA GGT GGG CCC TGG GCA GAA GTC AGA GAT TCT 960 GlyPro Val Cys Ala Ala Gly Gly Pro Trp Ala Glu Val Arg Asp Ser 305 310 315320 AGG ACG TTC ACA CTG GTC AGC GAG GTT GAG ACG CAA GGA GAC CTC TCG 1008Arg Thr Phe Thr Leu Val Ser Glu Val Glu Thr Gln Gly Asp Leu Ser 325 330335 AGG AAG ATT CCC ACA GAG GAT GAG TAC ACG GAC CGG CCC TCG CAG CCT 1056Arg Lys Ile Pro Thr Glu Asp Glu Tyr Thr Asp Arg Pro Ser Gln Pro 340 345350 TCG ACT GGT TCA CTG CTC CTA ATC CAG CAG GGA AGC AAA TCT ATA CCC 1104Ser Thr Gly Ser Leu Leu Leu Ile Gln Gln Gly Ser Lys Ser Ile Pro 355 360365 CCA TTC CAG GAG CCC CTG GAA GTG GGG GAG AAC GAC AGT TTA AGC CAG 1152Pro Phe Gln Glu Pro Leu Glu Val Gly Glu Asn Asp Ser Leu Ser Gln 370 375380 TGT TTC ACC GGG ACT GAA AGC ACG GTG GAT TCT GAG GGC TGT GAC TTC 1200Cys Phe Thr Gly Thr Glu Ser Thr Val Asp Ser Glu Gly Cys Asp Phe 385 390395 400 ACT GAG CCT CCG AGC AGA ACT GAC TCT ATG CCC GTG TCC CCT GAA AAG1248 Thr Glu Pro Pro Ser Arg Thr Asp Ser Met Pro Val Ser Pro Glu Lys 405410 415 CAC CTG ACA AAA GAA ATA GAA GGT GAC AGT TGC CTC CCC TGG GTG GTC1296 His Leu Thr Lys Glu Ile Glu Gly Asp Ser Cys Leu Pro Trp Val Val 420425 430 AGC TCC AAC TCA ACA GAT GGC TAC ACA GGC AGT GGG AAC ACT CCT GGG1344 Ser Ser Asn Ser Thr Asp Gly Tyr Thr Gly Ser Gly Asn Thr Pro Gly 435440 445 GAG GAC CAT GAA CCC TTT CCA GGG TCC CTG AAA TGT GGA CCA TTG CCC1392 Glu Asp His Glu Pro Phe Pro Gly Ser Leu Lys Cys Gly Pro Leu Pro 450455 460 CAG TGT GCC TAC AGC ATG GGC TTT CCC AGT GAA GCA GCA GCC AGC ATG1440 Gln Cys Ala Tyr Ser Met Gly Phe Pro Ser Glu Ala Ala Ala Ser Met 465470 475 480 GCA GAG GCG GGA GTA CGG CCC CAG GAC AGG GCT GAT GAG AGG GGAGCC 1488 Ala Glu Ala Gly Val Arg Pro Gln Asp Arg Ala Asp Glu Arg Gly Ala485 490 495 TCA GGG TCC GGG AGC TCC CCC AGT GAC CAG CCA CCT GCC TCT GGGAAC 1536 Ser Gly Ser Gly Ser Ser Pro Ser Asp Gln Pro Pro Ala Ser Gly Asn500 505 510 GTG ACT GGA AAC AGT AAC TCC ACG TTC ATC TCT AGC GGG CAG GTGATG 1584 Val Thr Gly Asn Ser Asn Ser Thr Phe Ile Ser Ser Gly Gln Val Met515 520 525 AAC TTC AAG GGT GAC ATC ATC GTG GTG TAT GTC AGC CAG ACC TCGCAG 1632 Asn Phe Lys Gly Asp Ile Ile Val Val Tyr Val Ser Gln Thr Ser Gln530 535 540 GAG GGC CCG GGT TCC GCA GAG CCC GAG TCG GAG CCC GTG GGC CGCCCT 1680 Glu Gly Pro Gly Ser Ala Glu Pro Glu Ser Glu Pro Val Gly Arg Pro545 550 555 560 GTG CAG GAG GAG ACG CTG GCA CAC AGA GAC TCC TTT GCG GGCACC GCG 1728 Val Gln Glu Glu Thr Leu Ala His Arg Asp Ser Phe Ala Gly ThrAla 565 570 575 CCG CGC TTC CCC GAC GTC TGT GCC ACC GGG GCT GGG CTG CAGGAG CAG 1776 Pro Arg Phe Pro Asp Val Cys Ala Thr Gly Ala Gly Leu Gln GluGln 580 585 590 GGG GCA CCC CGG CAG AAG GAC GGG ACA TCG CGG CCG GTG CAGGAG CAG 1824 Gly Ala Pro Arg Gln Lys Asp Gly Thr Ser Arg Pro Val Gln GluGln 595 600 605 GGT GGG GCG CAG ACT TCA CTC CAT ACC CAG GGG TCC GGA CAATGT GCA 1872 Gly Gly Ala Gln Thr Ser Leu His Thr Gln Gly Ser Gly Gln CysAla 610 615 620 GAA TGA 1878 Glu 625 625 amino acids amino acid linearprotein 15 Met Ala Pro Arg Ala Arg Arg Arg Arg Gln Leu Pro Ala Pro LeuLeu 1 5 10 15 Ala Leu Cys Val Leu Leu Val Pro Leu Gln Val Thr Leu GlnVal Thr 20 25 30 Pro Pro Cys Thr Gln Glu Arg His Tyr Glu His Leu Gly ArgCys Cys 35 40 45 Ser Arg Cys Glu Pro Gly Lys Tyr Leu Ser Ser Lys Cys ThrPro Thr 50 55 60 Ser Asp Ser Val Cys Leu Pro Cys Gly Pro Asp Glu Tyr LeuAsp Thr 65 70 75 80 Trp Asn Glu Glu Asp Lys Cys Leu Leu His Lys Val CysAsp Ala Gly 85 90 95 Lys Ala Leu Val Ala Val Asp Pro Gly Asn His Thr AlaPro Arg Arg 100 105 110 Cys Ala Cys Thr Ala Gly Tyr His Trp Asn Ser AspCys Glu Cys Cys 115 120 125 Arg Arg Asn Thr Glu Cys Ala Pro Gly Phe GlyAla Gln His Pro Leu 130 135 140 Gln Leu Asn Lys Asp Thr Val Cys Thr ProCys Leu Leu Gly Phe Phe 145 150 155 160 Ser Asp Val Phe Ser Ser Thr AspLys Cys Lys Pro Trp Thr Asn Cys 165 170 175 Thr Leu Leu Gly Lys Leu GluAla His Gln Gly Thr Thr Glu Ser Asp 180 185 190 Val Val Cys Ser Ser SerMet Thr Leu Arg Arg Pro Pro Lys Glu Ala 195 200 205 Gln Ala Tyr Leu ProSer Leu Ile Val Leu Leu Leu Phe Ile Ser Val 210 215 220 Val Val Val AlaAla Ile Ile Phe Gly Val Tyr Tyr Arg Lys Gly Gly 225 230 235 240 Lys AlaLeu Thr Ala Asn Leu Trp Asn Trp Val Asn Asp Ala Cys Ser 245 250 255 SerLeu Ser Gly Asn Lys Glu Ser Ser Gly Asp Arg Cys Ala Gly Ser 260 265 270His Ser Ala Thr Ser Ser Gln Gln Glu Val Cys Glu Gly Ile Leu Leu 275 280285 Met Thr Arg Glu Glu Lys Met Val Pro Glu Asp Gly Ala Gly Val Cys 290295 300 Gly Pro Val Cys Ala Ala Gly Gly Pro Trp Ala Glu Val Arg Asp Ser305 310 315 320 Arg Thr Phe Thr Leu Val Ser Glu Val Glu Thr Gln Gly AspLeu Ser 325 330 335 Arg Lys Ile Pro Thr Glu Asp Glu Tyr Thr Asp Arg ProSer Gln Pro 340 345 350 Ser Thr Gly Ser Leu Leu Leu Ile Gln Gln Gly SerLys Ser Ile Pro 355 360 365 Pro Phe Gln Glu Pro Leu Glu Val Gly Glu AsnAsp Ser Leu Ser Gln 370 375 380 Cys Phe Thr Gly Thr Glu Ser Thr Val AspSer Glu Gly Cys Asp Phe 385 390 395 400 Thr Glu Pro Pro Ser Arg Thr AspSer Met Pro Val Ser Pro Glu Lys 405 410 415 His Leu Thr Lys Glu Ile GluGly Asp Ser Cys Leu Pro Trp Val Val 420 425 430 Ser Ser Asn Ser Thr AspGly Tyr Thr Gly Ser Gly Asn Thr Pro Gly 435 440 445 Glu Asp His Glu ProPhe Pro Gly Ser Leu Lys Cys Gly Pro Leu Pro 450 455 460 Gln Cys Ala TyrSer Met Gly Phe Pro Ser Glu Ala Ala Ala Ser Met 465 470 475 480 Ala GluAla Gly Val Arg Pro Gln Asp Arg Ala Asp Glu Arg Gly Ala 485 490 495 SerGly Ser Gly Ser Ser Pro Ser Asp Gln Pro Pro Ala Ser Gly Asn 500 505 510Val Thr Gly Asn Ser Asn Ser Thr Phe Ile Ser Ser Gly Gln Val Met 515 520525 Asn Phe Lys Gly Asp Ile Ile Val Val Tyr Val Ser Gln Thr Ser Gln 530535 540 Glu Gly Pro Gly Ser Ala Glu Pro Glu Ser Glu Pro Val Gly Arg Pro545 550 555 560 Val Gln Glu Glu Thr Leu Ala His Arg Asp Ser Phe Ala GlyThr Ala 565 570 575 Pro Arg Phe Pro Asp Val Cys Ala Thr Gly Ala Gly LeuGln Glu Gln 580 585 590 Gly Ala Pro Arg Gln Lys Asp Gly Thr Ser Arg ProVal Gln Glu Gln 595 600 605 Gly Gly Ala Gln Thr Ser Leu His Thr Gln GlySer Gly Gln Cys Ala 610 615 620 Glu 625 20 amino acids amino acid linearprotein 16 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp ValPro 1 5 10 15 Gly Ser Thr Gly 20 5 amino acids amino acid linear protein17 Asp Tyr Lys Asp Glu 5 6 amino acids amino acid linear protein 18 HisHis His His His His 5 33 amino acids amino acid linear protein 19 ArgMet Lys Gln Ile Glu Asp Lys Ile Glu Glu Ile Leu Ser Lys Ile 1 5 10 15Tyr His Ile Glu Asn Glu Ile Ala Arg Ile Lys Lys Leu Ile Gly Glu 20 25 30Arg

We claim:
 1. An isolated DNA selected from the group consisting of: (a)a DNA encoding a protein having an amino acid sequence as set forth inSEQ ID NO:6, wherein the protein has an amino terminus selected from thegroup consisting of an amino acid between amino acid 1 and amino acid33, inclusive, of SEQ ID NO:6, and a carboxy terminus selected from thegroup consisting an amino acid between amino acid 196 and amino acid616, inclusive; (b) a DNA encoding a protein having an amino acidsequence as set forth in SEQ ID NO:15, wherein the protein has an aminoterminus selected from the group consisting of an amino acid betweenamino acid 1 and amino acid 30, inclusive, of SEQ ID NO:15, and acarboxy terminus selected from the group consisting an amino acidbetween amino acid 197 and amino acid 625, inclusive; (c) DNA moleculescapable of hybridization to the DNA of (a) or (b) under stringentconditions, and which encode biologically active RANK; and (d) DNAmolecules encoding fragments of proteins encoded by the DNA of (a), (b)or (c).
 2. The isolated DNA of claim 1, which encods a RANK polypeptidethat is at least about 80% identical in amino acid sequence to thenative form of RANK
 3. The isolated DNA of claim 1, which encodes asoluble RANK polypeptide.
 4. The isolated DNA of claim 2, which encodesa soluble RANK polypeptide.
 5. The isolated DNA of claim 3, whichfurther comprises a DNA encoding a polypeptide selected from the groupconsisting of an immunoglobulin Fc domain, an immnunoglobulin Fc mutein,a FLAG^(™) tag, a peptide comprising at least about 6 His residues, aleucine zipper, and combinations thereof.
 6. The isolated DNA of claim4, which further comprises a DNA encoding a polypeptide selected fromthe group consisting of an immunoglobulin Fc domain, an immunoglobulinFc mutein, a FLAG^(™) tag, a peptide comprising at least about 6 Hisresidues, a leucine zipper, and combinations thereof.
 7. A recombinantexpression vector comprising a DNA sequence according to claim
 1. 8. Arecombinant expression vector comprising a DNA sequence according toclaim
 2. 9. A recombinant expression vector comprising a DNA sequenceaccording to claim
 3. 10. A recombinant expression vector comprising aDNA sequence according to claim
 4. 11. A recombinant expression vectorcomprising a DNA sequence according to claim
 5. 12. A recombinantexpression vector comprising a DNA sequence according to claim 6 .
 13. Ahost cell transformed or transfected with an expression vector accordingto claim
 7. 14. A host cell transformed or transfected with anexpression vector according to claim
 8. 15. A host cell transformed ortransfected with an expression vector according to claim
 9. 16. A hostcell transformed or transfected with an expression vector according toclaim
 10. 17. A host cell transformed or transfected with an expressionvector according to claim
 11. 18. A host cell transformed or transfectedwith an expression vector according to claim
 12. 19. A process forpreparing a RANK protein, comprising culturing a host cell according toclaim 13 under conditions promoting expression and recovering the RANK.20. A process for preparing a RANK protein, comprising culturing a hostcell according to claim 14 under conditions promoting expression andrecovering the RANK.
 21. A process for preparing a RANK protein,comprising culturing a host cell according to claim 15 under conditionspromoting expression and recovering the RANK.
 22. A process forpreparing a RANK protein, comprising culturing a host cell according toclaim 16 under conditions promoting expression and recovering the RANK.23. A process for preparing a RANK protein, comprising culturing a hostcell according to claim 17 under conditions promoting expression andrecovering the RANK.
 24. A process for preparing a RANK protein,comprising culturing a host cell according to claim 18 under conditionspromoting expression and recovering the RANK.
 25. An isolated DNAselected from the group consisting of oligonucleotides of at least about17 nucleotides in length, oligonucleotides of at least about 25nucleotides in length, and oligonucleotides of at least about 30nucleotides in length, which is a fragment of the DNA of SEQ ID NO:5 orSEQ ID NO:14.
 26. An isolated RANK polypeptide selected from the groupconsisting of: (a) a polypeptide having an amino acid sequence of aminoacids 33 through 196 of SEQ ID NO: 6; (b) a polypeptide having an aminoacid sequence of amino acids 30 through 197 of SEQ ID NO: 15; (c) a RANKpolypeptide encoded by a DNA capable of hybridization to a DNA encodingthe protein of (a) or (b) under stringent conditions, and which isbiologically active; and (d) fragments of the polypeptides of (a), (b)or (c) which are biologically active.
 27. The protein according to claim26, having an amino acid sequence at least about 80% identical to SEQ IDNO:6 or SEQ ID NO:15.
 28. The protein according to claim 27, which is asoluble RANK.
 29. The protein according to claim 26, which is a solubleRANK.
 30. A soluble RANK protein which further comprises a peptideselected from the group consisting of an immunoglobulin Fc domain, animmunoglobulin Fc mutein, a FLAG^(™) tag, a peptide comprising at leastabout 6 His residues, a leucine zipper, and combinations thereof.
 31. Anantibody immunoreactive with RANK polypeptide according to claim
 26. 32.The antibody according to claim 31, which is a monoclonal antibody. 33.A method of inhibiting activation of NFκB, comprising contacting a cellthat expresses membrane-associated RANK with a soluble RANK and allowingthe soluble RANK to bind RANKL and inhibit binding thereof to the cell.34. A method of regulating an immune or inflammatory response,comprising administering a soluble RANK polypeptide composition to anindividual at risk for an immune or inflammatory response, and allowingthe soluble RANK to bind RANKL and inhibit binding thereof to cellsexpressing RANK.